Supplemental Materials and Methods Isolation of liposarcoma … · 2015-07-24 · Lipo029b Female 46 Recurrent RPS WDLPS 61 Lipo445a Female 66 Primary RPS WDLPS 90 Lipo863b Male 73

Supplemental Materials and Methods

Isolation of liposarcoma cells and establishment of liposarcoma cell lines/strains

This procedure was conducted with approval from the Institutional Review Board at The

University of Texas M.D. Anderson Cancer Center and patients’ informed consent. Tumor cell

isolation was conducted as previously described. Each of the 7 cell lines/strains used in this

study were derived from tumors independent of those represented on the liposarcoma TMA.

Lipo224a, 224b, 247b, 445a, and 863b were derived from WDLPS and DDLPS tumors obtained

from patients independent of those represented on the liposarcoma TMA. Briefly, fresh sterile

samples from surgically resected tumors were minced in culture medium and digested via

incubation with collagenase type I (3%), DNase I (0.02%), and hyaluronidase (1.5mg/mL) at

37oC for 2-4 h. Samples were strained through wire mesh screens and undigested tissue was

discarded. After centrifugation, washes, and resuspension in PBS, samples were gently

transferred to Histopaque tubes containing 10mL Histopaque (100%, Sigma) overlayed with

15mL of Histopaque (75%). The tubes were then centrifuged at 4oC for 30 min at 1200 g. After

centrifugation, tumor cells located in the top interface (over the 75% Ficoll) were collected and

plated (high fat containing cells have been discarded). Cells were cultured and passaged in

DMEM supplemented with 10% FBS.

MDM2 FISH analysis

FISH was performed on fixed cultured cells with a laboratory-developed BAC probe

cocktail using BAC clones RP11- RP11-185H13, RP11-450G15, RP11-816C9, RP11-630N19,

RP11-717F7, RP11-1104N20 and RP11-426B12 (purchased from the Children’s Hospital

Oakland research Institute, Oakland, CA, USA) specific for the 12q15 region (spectrum orange)

and a probe specific for the centromeric region of chromosome 12 (spectrum green; Abbott

Molecular, DesPlaines, IL, USA). A minimum of 100 nuclei per slide were analyzed. The

average number of MDM2 and CEP12 signals was then determined and a MDM2/CEP12 ratio

was calculated for each case. A ratio ≥2 was considered amplified for the MDM2 gene,

whereas a ratio <2.0 was considered nonamplified. A ratio of <2.0 with >2 signals of both

probes was considered polysomic for CEP12. The established DDLPS cell line LPS-141 and

normal adipocytes were used as positive and negative controls, respectively.

Variable n (% or range) WD (n=101) DD (n=50) p-value

Median Age at Initial Surgery 60.5 (22.3-85.5) 60.2 (22.3-85.5) 61.1 (34.7-82.4) 0.404

Gender 0.086

Female 60 (39.7) 45 (44.6) 15 (30)

Male 91 (60.3) 56 (55.4) 35 (70)

Location of Initial Surgery 0.633

MDACC 141 (93.4) 95 (94.1) 46 (92)

Other Institution 10 (6.6) 6 (5.9) 4 (8)

Ethnicity 0.191

White 118 (78.1) 76 (75.2) 42 (84)

Black 8 (5.3) 5 (5) 3 (6)

Hispanic 20 (13.2) 16 (15.8) 4 (8)

Asian 5 (3.3) 4 (4) 1 (2)

Median Tumor Size (cm) 24.25 (2.5-100) 24.25 (2.5-100) 24 (4.5-68) 0.948

Primary Tumor Histology

WD 101 (66.9) - - -

DD or WD and DD 50 (33.1) - - -

Tumor Site <0.001

Retroperitoneum/abdomen/pelvis 109 (72.2) 61 (60.4) 48 (96)

Extremity 33 (21.9) 33 (32.7) 0 (0)

Buttock 5 (3.3) 4 (4) 1 (2)

Inguinal, scrotal only 2 (1.3) 1 (1) 1 (2)

Chest/mediastinum 1 (0.7) 1 (1) 0 (0)

Chest/abdominal wall 1 (0.7) 1 (1) 0 (0)

Multifocality 0.007

Unifocal 123 (81.5) 88 (87.1) 35 (70)

Multifocal 27 (17.9) 12 (11.9) 15 (30)

Unknown 1 (0.7) 1 (1) 0 (0)

Completeness of Resection 0.046

R0/R1 (Macroscopically Complete) 137 (90.7) 95 (94.1) 42 (84)

R2 (Macroscopically Incomplete) 14 (9.3) 6 (5.9) 8 (16)

Tumor Integrity 0.615

Intact Tumor 136 (90.1) 93 (92.1) 43 (86)

Tumor Rupture or Transection 13 (8.6) 8 (7.9) 5 (10)

Unknown 2 (1.3) 0 (0) 2 (4)

Chemotherapy <0.001

No 127 (84.1) 94 (93.1) 33 (66)

Yes 23 (15.2) 6 (5.9) 17 (34)

Unknown 1 (0.7) 1 (1) 0 (0)

Radiation therapy 0.863

No 131 (86.8) 87 (86.1) 44 (88)

Yes 19 (12.6) 13 (12.9) 6 (12)

Unknown 1 (0.7) 1 (1) 0 (0)

Number of Recurrences 0.027

0 76 (50.3) 58 (57.4) 18 (36)

1 40 (26.5) 23 (22.8) 17 (24)

2 17 (11.3) 9 (8.9) 8 (16)

3 14 (9.3) 7 (6.9) 7 (14)

4 or more 4 (2.7) 4 (4) 0 (0)

Distant metastasis 18 (11.9) 7 (6.9) 11 (22) 0.007

Median Length of Followup (months) 75.37 (0.33-236.77) 91.1 (6.3-236.77) 37.58 (0.33-158.63) <0.001

Status at Last Followup <0.001

Dead of Disease 47 (31.1) 18 (17.8) 29 (58)

Dead of Other Cause/Cause Unknown 9 (6) 9 (8.9) 12 (24)

Alive with Disease 33 (21.9) 21 (20.8) 9 (18)

Alive No Evidence of Disease 62 (41.1) 53 (52.5) 0 (0)

Last Known Status <0.001

Dead of Disease 62 (41.1) 24 (23.8) 38 (76)

Dead of Other Cause/Cause Unknown 16 (10.6) 15 (14.9) 1 (2)

Alive with Disease 18 (11.9) 145 (14.9) 3 (6)

Alive No Evidence of Disease 55 (36.5) 46 (45.5) 8 (16)

5 year Overall Survival (%) 62.8 77.0 33.0

Median Overall Survival (months) 88.97 (6.3-244.5) 97.43 (6.3-244.5) 40 (6.87-158.63) <0.001

Median Local Recurrence-Free Survival (months) 65.33 (2.33-243.27) 91.83 (2.53-243.27) 14 (2.33-220) <0.001

Median Distant Recurrence-Free Survival (months) 252.83 (1.63-244.5) 120.8 (31.07-277.77) 99.97 (1.23-283.9) <0.035

Supp Figure 1

Supplemental Figure 1. Clinicopathologic characteristics of patients with primary WDLPS and DDLPS represented

on tissue microarray (n=151). Discrete data were analyzed using a Fisher exact comparison, and continuous data were

analyzed using a t test.

SuppFigure 2

A 0

1

2

3

0

1

2

3

B

Supplemental Figure 2. H3K9me3 is differentially expressed in WDLPS and DDLPS tumors. (A) Representative photographs of immunostained WDLPS from TMA scored by

staining intensity (0=none, 1=low, 2=medium, 3=high) for H3K9me3. (B) Representative photographs of immunostained DDLPS from TMA scored by staining intensity (0=none, 1=low,

2=medium, 3=high) for H3K9me3.

200x 200x

200x 200x

200x 200x

200x 200x

A

B

C

224a 224b 863b

247b

246

029b 445a

Designated

Name

Patient

Age

Patient

Gender

Primary/Recurrent

Tumor

Tumor

Location Histology

% Cells with

MDM2

Amplification

Lipo224a Female 81 Primary Pelvis DDLPS 98

Lipo224b Female 81 Recurrent Pelvis DDLPS 98

Lipo246 Male 57 Recurrent RPS DDLPS 99

Lipo247b Male 67 Recurrent RPS WDLPS 64

Lipo029b Female 46 Recurrent RPS WDLPS 61

Lipo445a Female 66 Primary RPS WDLPS 90

Lipo863b Male 73 Recurrent RPS DDLPS 91

224a 224b 863b

247b

246

029b 445a

Supp Figure 3

Supplemental Figure 3. WDLPS and DDLPS cell lines. (A) Characteristics of WDLPS and DDLPS cell lines and tumors from which cell

lines were derived. (B) H+E staining of original tissue/tumor from which cell lines were derived. (C) Confirmation of MDM2 amplification by

FISH analysis.

100x 100x

100x 100x

100x 100x

100x

B C

D E

0.1

0.2

0.3

0.4

Genomic Region (5' −> 3')

Rea

d c

ou

nt P

er

Mill

ion m

app

ed r

eads

−5000 5'End 33% 66% 3'End 5000

224a (DD)

224b (DD)

247b (WD)

029b (WD)

0.1

0.2

0.3

0.4

0.5

0.6

Genomic Region (5' −> 3')

Rea

d c

ou

nt P

er

Mill

ion m

app

ed r

eads

−5000 5'End 33% 66% 3'End 5000

224a (DD)

224b (DD)

247b (WD)

029b (WD)

Supp Figure 4

Supplemental Figure 4. Number of H3K9me3 regions differentially enriched among WDLPS and DDLPS cell lines. (A) Overview and

quality metrics of ChIP-sequencing and data analysis. (B) Number of regions preferentially enriched in DDLPS cell lines. (C) Number of regions

preferentially enriched in WDLPS cell lines. (D) Aggregate plots showing profiles of H3K9me3 around identified peaks specific to DDLPS cell

lines. (E) Aggregate plots showing profiles of H3K9me3 around identified peaks specific to WDLPS cell lines.

A

Supp Figure 5

Supplemental Figure 5. Heatmap of differentially expressed genes

among DDLPS and WDLPS cell lines on microarray analysis. Each

sample performed in triplicate.

A

B

224a (DD)

224b (DD)

246 (DD)

863b (DD)

029b (WD)

247b (WD)

445a (WD)

all DDLPS cell lines all WDLPS cell lines

Supp Figure 6

Supplemental Figure 6. KLF6 is epigenetically silenced in DDLPS. (A) Histograms of ChIP

fragments across the KLF6 locus for each LPS cell line. H3K9me3 is enriched at the regulatory

region of KLF6 in multiple DDLPS cell lines compared to WDLPS cell lines. Each track is normalized

to 10 million reads with same track height and vertical viewing range. (B) Box plot showing KLF6

expression in DDLPS cell lines (224a, 224b, 246, 863b) compared to WDLPS (029b, 247b, 445a)

cell lines by microarray analysis (n=3).

A

B

Supp Figure 7

Re

lati

ve K

LF6

-wt

exp

ress

ion

(m

RN

A)

DD Normal Fat WD

Re

lati

ve K

LF6

-sv1

exp

ress

ion

(m

RN

A)

DD Normal Fat WD

Supplemental Figure 7. Wild type KLF6 (KLF6-wt) and KLF6 splice variant 1 (KLF6-sv1)

are underexpressed in human DDLPS tumors compared to WDLPS tumors. (A) KLF6-wt

expression among human DDLPS tumors (n=17) compared to WDLPS tumors (n=13) and

normal fat (n=6) assessed by RT-qPCR normalized to GAPDH (mean +/- SD; n=3). (B) KLF6-

sv1 expression among human DDLPS tumors (n=17) compared to WDLPS tumors (n=13) and

normal fat (n=6) assessed by RT-qPCR normalized to GAPDH (mean +/- SD; n=3). Mann-

Whitney U and Kruskal-Wallis tests were used. These data are representative of 2

experiments.

Supplemental Figure 8. Relative KLF6 copy number in WDLPS and DDLPS cell lines and tumors. (A) Relative KLF6 copy number of WDLPS (n=3) and DDLPS (n=4) cell lines assessed by

Taqman copy number variation assay (RNaseP reference) (mean +/- SD; n=3). (B) Relative KLF6 copy number of WDLPS (n=15) and DDLPS (n=19) tumors assessed by Taqman copy number

variation assay (RNaseP reference) (mean +/- SD; n=3). (C) Relative KLF6 copy number and protein expression among normal fat, WDLPS, and DDLPS samples show poor correlation between

copy number and protein expression between and within tissue/tumor types. These data are representative of 2 experiments.

C

Sample Specimen Histology Relative KLF6 Gene Copy

Number Relative KLF6 Protein Expression1

N58 Normal fat 3.1 33.95




N51 Normal fat 2.0 Below detection


T68 DD 2.8 0.65

T88 DD 2.8 Below detection

T50b DD 2.6 Below detection

T61b DD 2.3 9.59

T71 DD 2.3 0.14

T7 DD 2.2 0.80

T65 DD 2.2 1.08


T9a DD 2.0 28.21

T64 DD 2.0 0.15

T80 DD 1.9 0.88


T83 DD 1.8 86.56

T25 DD 1.7 1.00

T67b DD 1.7 0.07

T12a DD 1.6 1.08

T33 DD 1.6 0.28

T90 DD 1.3 2.34


T47 WD 3.1 23.13

T53 WD 3.1 17.29

T85 WD 2.9 1.58

T43b WD 2.8 4.19

T86 WD 2.8 80.59

T63 WD 2.7 5.38

T66a WD 2.7 10.94

T51 WD 2.4 16.72

T20 WD 2.2 179.17

T28 WD 2.0 7.51

T9b WD 1.9 5.71

T76b WD 1.9 Below detection

T12b WD 1.8 11.35

T36 WD 1.2 76.59

1Relative to sample T25 A

B

Re

lati

ve K

LF6

co

py

nu

mb

er

Re

lati

ve K

LF6

co

py

nu

mb

er

Supp Figure 8

A B C

F

SuppFigure 9 R

ela

tive

KLF

6 e

xpre

ssio

n (m

RN

A)

Control KLF6

Control

KLF6

p < 0.001

KLF

6

Co

ntr

ol

D

% C

on

flu

en

ce

Hours

KLF

6

C

on

tro

l

G

Control

KLF6

* p<0.05

Re

lati

ve e

xpre

ssio

n (

mR

NA

)

CEBPA CEBPB PPARG

Supplemental Figure 9. KLF6 overexpression inhibits DDLPS cell proliferation, induces expression of adipogenesis differentiation

markers, and results in cellular senescence. (A) KLF6 overexpression in DDLPS cell line 224b assessed by RT-qPCR normalized to GAPDH

(mean +/- SD; n=3). Student’s t test. (B) Cellular proliferation of DDLPS cell line 224b overexpressing KLF6 versus control (mean +/- SEM; n=10).

Mann-Whitney U test. (C) Image of invasive cells from Boyden chamber assay on DDLPS cell line 224b cells stably expressing KLF6 or control.

(D) Quantification of invaded cells from Boyden chamber assay in DDLPS 224b cells expressing control or KLF6. Plot shows relative absorbance

from crystal violet stain of invaded cells (mean +/- SEM; n=3). (E) Image of invasive cells from Boyden chamber assay on DDLPS 246 cell line

cells stably expressing KLF6 or control. (F) Image of invasive cells from Boyden chamber assay on DDLPS 224a cell line cells stably expressing

KLF6 or control. (G) B-galactosidase staining assaying for cellular senescence in DDLPS cell line 224b overexpressing KLF6 versus control. (H)

Relative expression of adipogenesis associated markers CEBPA, CEBPB and PPARG in DDLPS cell line 224b overexpressing KLF6 versus

control as assessed by RT-qPCR normalized to GAPDH (mean +/- SD; n=3). Student’s t test. These data are representative of 2 experiments.

E

H

KLF

6

Co

ntr

ol

KLF

6

Co

ntr

ol

DDLPS 246 DDLPS 224a

DDLPS 224b

0 .0 0

0 .0 5

0 .1 0

0 .1 5

0 .2 0

0 .2 5

Re

lati

ve A

bso

rban

ce

(Cry

stal

Vio

let,

59

0n

M) DDLPS 224b

Control KLF6

DDLPS 224b DDLPS 224b

Supplemental Figure 10. Chaetocin, an inhibitor of SUV39H1, inhibits DDLPS proliferation, inhibits H3K9 trimethylation globally and at the KLF6 locus, and induces upregulation of

KLF6 and proadipogenesis factors PPARG, CEBPA, and CEBPB. (A) Proliferation of DDLPS cell line 224b upon chaetocin treatment (mean +/- SEM; n=6). (B) Global levels of H3K9me3 by

western blot analysis in DDLPS cell line 224b following chaetocin treatment (30nM). 2-way ANOVA test. (C) KLF6 expression by RT-qPCR analysis in DDLPS cell line 224b following chaetocin

treatment (30nM) normalized to GAPDH (mean +/- SD, n=3). (D) ChIP-qPCR analysis of H3K9me3 enrichment at the KLF6 locus in DDLPS 224b cells treated with vehicle vs chaetocin 30nM for

24hrs (mean +/- SD, n=3). Student’s t test. (E) Relative expression of PPARG, CEBPA, and CEBPB by RT-qPCR in DDLPS cell line 224b following chaetocin treatment (30nM) normalized to

GAPDH (mean +/- SD, n=3). Student’s t test. These data are representative of 2 experiments.

B A

% C

on

flu

en

ce

Co

ntr

ol

24

hrs

H3

H3K9me3

Hours Following Chaetocin Treatment

p < 0.001

C

Control 24hrs 48hrs Re

lati

ve K

LF6

exp

ress

ion

(m

RN

A)

D

Re

lati

ve e

xpre

ssio

n (

mR

NA

)

CEBPA CEBPB PPARG

E

SuppFigure 10

Supp Figure 11

Supplemental Figure 11. EC50 determination of chaetocin in DDLPS cell line 246

Re

lati

ve S

UV

39

H1

exp

ress

ion

A B

Supp Figure 12

Re

lati

ve S

UV

39

H1

exp

ress

ion

(mR

NA

)

DD Normal Fat WD

Supplemental Figure 12. DDLPS and WDLPS do not differ in SUV39H1 gene expression. (A) Relative SUV39H1 gene

expression among DDLPS and WDLPS cell lines by RT-qPCR analysis normalized to GAPDH (mean +/- SD, n=3). (B-K)

Relative mRNA expression of SUV39H1 (B, Mann-Whitney U and Kruskal-Wallis tests), SUV39H2, (C), SETDB1 (D), SETDB2

(E), EHMT1 (F), EHMT2 (G), JMJD2A (H, Student’s t test), JMJD2B (I), JMJD2C (J) and JMJD2D (K) among DDLPS and

WDLPS tumors by RT-qPCR analysis normalized to GAPDH (mean +/- SD, n=3). These data are representative of 2

experiments.

DD Normal Fat WD DD Normal Fat WD

JMJD2B JMJD2C JMJD2D I J K

P =0.045

P =0.037

DD Normal Fat WD


EHMT1 EHMT2 JMJD2A F G H

DD Normal Fat WD


SUV39H2 SETDB1 SETDB2 C D E

DD Normal Fat WD

Documents

Supplemental Materials and Methods Isolation of liposarcoma … · 2015-07-24 · Lipo029b Female 46 Recurrent RPS WDLPS 61 Lipo445a Female 66 Primary RPS WDLPS 90 Lipo863b Male 73