Adipose derived stem cells in Adipose derived stem cells in endometrial cancers Ann Klopp, MD PhD Advances in Endometrial Cancer Epidemiology and Biology March 18 th , 2014
Tumor Stroma Where does it come from?
Bone marrow origin of tumor Bone marrow origin of tumor stroma stroma β -gal Tg RAG-1 -/- , H-2b / BMT XRT Pancreas SCID Ca S.C. H-2d 4 wks 2-4 wks 40% of the myofibroblasts in the cancer-induced stroma were of BM origin at 4 f X-gal weeks. Ishii et al, BBRC, 309:232, 2003
MSC/ASC MSC/ASC Therapeutic Immune modulation for Marrow stromal cells/ hematopoetic transplantion Adipose stromal cells Tissue engineering Tissue engineering Mesenchymal stem cells Definition Plastic adherent l dh Resident and circulating Mesenchymal markers Bone marrow Multipotent Fat Characteristics Endogenous functions g Cells can can be readily, s c c b d y, Support hematopoesis expanded and restransplanted. Wound repair Migrate to sites of injury and tumors
Bone-marrow derived MSC engraft in MDA231 Breast Metastasis Murine Lungs with MDA231 metastasis g Normal Murine Lungs 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 which form tumor which form tumor stroma hi h f hi h f t t stroma t Zhang, Kolonin Cancer Research 2009
Kidd Plos 2012
Does obesity increase ASC Does obesity increase ASC engraftment? engraftment? f f ? ? Tumors in obese mice contain more ASC marker positive cells Tumors in obese mice contain more ASC marker positive cells Zhang, Kolonin Cancer Research 2012
Hypothesis: visceral adipose tissue Hypothesis: visceral adipose tissue provides provides ASC to id id ASC ASC ASC to intraperitoneal intraperitoneal i i i i l l tumors tumors tumors tumors
Visceral adiposity vs. BMI p y
Visceral adiposity and endometrial Visceral adiposity and endometrial cancer risk in post-menopausal women Waist to hip ratio Waist to hip ratio RR RR < 0.74 1.0 0.74-0.78 0 74 0 78 1 09 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- Rare metastasis abdominal cancers Increases hypergycemia, Less metabolic effects on insulin hyperinsulinemia, hyperTG, impaired resistance glucose tolerance Drains through the liver g Venous drainage through systemic veins g g y Larger adipocytes (more insulin Smaller adipocytes (higher uptake of resistance) FFA and TG) Well vascularized and innervated Less well vascularized and innervated More inflammatory cell infiltrating y g Less inflammatory cell infiltrate y Higher expression of pro-inflammatory Less inflammatory cytokines cytokines (TNF-alpha, CRP, IL-6) Accumulation supported by lower Accumulation promoted by estrogen estrogen
Specimen Specimen Diagnosis Diagnosis Omentum Omentum BMI BMI Stage Stage involved OSC1 AdenoCA of - 25 Recurrent endometrium and ovary OSC2 Granulosa cell tumor of + 31.4 Recurrent the ovary sc ASC sc-ASC lipoaspirate lipoaspirate n/a n/a unknown unknown n/a n/a BM-MSC Normal bone marrow n/a unknown n/a donors 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. subcutaneous) change the ASC phenotype? b ) h h h ? • 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 with Is ASC population abnormal in patients with metastatic cancer?
Mince omentum Enzymatic digestion (collagenase and dispase) for 1 hour on shaker at 37 degrees for 1 hour on shaker at 37 degrees Centrifuge and resuspend in DMEM/ 10% FBS Centrifuge and resuspend in DMEM/ 10% FBS Filter through 100 µ m and 40 µ m Nylon cell strainer Centrifuge and resuspend pellet in red cell lysis buffer Incubate in 37 degree water bath I b t i 37 d t b th Count and plate cells Count and plate cells
In-vivo effects of OSC on In vivo effects of OSC on endometrial xenograft formation 1.6 x 6 10 4 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 xenografts Periphery Periphery 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 maturity O 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 endometrial xenografts by supporting tumor vasculature. Q Questions i • Could differences in O-ASC and sc-ASC be attributed to isolation techniques?
Omental vs. subcutaneous derived ASC: P i Paired clinical samples d li i l l Flouresence Flouresence F F Days after 300uM carboplatin y p Days Days
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 develop C57 mice fed high fat diet develop obesity and metabolic syndrome Injection 50 Lean Mice Obese Mice Obese Mice 40 ht (g) 30 Weig 20 10 0 0 50 100 150 132 Days
Adipos e density *** 10 10 *** 8 cm ^3) 6 Volum e (c 4 2 0 Le-SC Le-V Ob-SC Ob-V
ID8 ovarian cancers grow more rapidly ID8 ovarian cancers grow more rapidly in mice with diet-induced obesity t] lean lean [tota l c oun 2000000 obese 1500000 ne s c e nc e 1000000 500000 lum i 0 0 2 4 6 8 10 12 days
ASC isolation Lean Obese L-V- SubQ ASC Viscera l
Le-V-ASC Ob-V-ASC Le-SC-ASC Ob-SC-ASC CD29 CD34 CD31 CD11b 32
Ob-V-ASC Le-V-ASC Le-SC-ASC Ob-SC-ASC CD73 CD90 CD105 CD45 33
ASC proliferation ASC proliferation 2000000 Le-SC-ASC Le SC ASC Le-V-ASC 1500000 umber Ob-SC-ASC 1000000 1000000 cell nu Ob-V-ASC 500000 0 0 5 10 15 days
In-vitro adipocyte differentiation p y SubQ Visceral Lean Obese adipogenesis( total OD) 100000 ** ** 80000 sity tical Den 60000 ** 40000 p O 20000 0 Lean-SC-ASC Ob-SC-ASC Lean-V-ASC Ob-V-ASC
In-vitro osteocyte differentiation y SubQ Visceral Lean Obese osteogenesis(total OD) 600000 ** ** sity 400000 tical Den 200000 p p O 0 C C C C S S S S A A A A - - - - C C V V S S - - n b - - n a O b e a O L e L
SubQ Visceral Obese Obese Lean Lean Lean Obese P=0.05 P=0.321 Lean Obese
SubQ Visceral Lean Obese Ki67 Vessel Nuclear Ki67 in tumor 4+ pixels ** 10 * 30 ** 8 ent GSL I isolectin B4 percent Ki67 + pixels 20 6 4 10 2 p 0 0 perce C C C C 0 S S S S A A A A Le-SC Ob-SC Le-V Ob-V - - - - C C V V S S - - e b - - L O e b L O groups
SubQ Visceral Lean Obese Perilipin Vessel Nuclear Perilipin (pixel) ** 4 els ercent perilipin+ pixe 3 2 1 pe 0 Le-SC-ASC Ob-SC-ASC Le-V-ASC Ob-V-ASC groups
Analysis of malignant ascities ascites Macrophage in ascites 5 40 * 4 me (ml) 30 3 t cen volum erc 20 20 2 p 10 1 0 0 C C C C C C C C C C C C C C C C S S S S S S S S A A A A A A A A - - - - C C V V - - - - C C V V S - S - e b S S - - - - e L O b e b - - L O L e b O L O
SubQ Visceral F4/80 Vessel Nuclear Lean Lean Obese Ob-V-ASC *** 10 * * * 4/80+ pixels 8 6 percent F4 4 2 0 Le-SC Ob-SC Le-V Ob-V
MSC increase MCF-7 mammosphere formation MSC 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 250 ** ** SubQ Visceral 200 numbers 150 100 Lean 50 0 Le-SC-CM Ob-SC-CM Le-V-CM Ob-V-CM * * 200 150 m) Obese size(u 100 50 0 Le-SC-CM Le-V-CM Ob-SC-CM Ob-V-CM Le-SC-ctrl Le-V-ctrl Ob-SC-ctrl Ob-V-ctrl
ID8 spheroid formation in ASC-CM SubQ Visceral * 250 200 rs 150 e Lean b m u 100 n 50 0 C C V V l r S S - - t e b c - - L O e b L O 250 200 Obese ) m 150 (u e iz 100 s 50 0 C C V V Le-V- l Ob-V- r S S - - t e b c - - L O e b L O ASC ASC
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