supplementary methods, figures 1-10

1

Supplementary Methods MTT Proliferation Assay: 1x103 cells were plated in triplicate and were treated with

DMSO vehicle control or increasing concentrations of U0126 (0.1 µM, 1 µM, 10 µM,

and 20 µM from Promega). After 24 or 48 hours of incubation, the Vybrant MTT cell

Proliferation Assay Kit (Invitrogen) was used per the manufacturer’s protocol. For

assessment of effects of CD44 knockdown on MOC2 and 10 cells, 6000 cells of each

specific cell line were plated in 96-well plates and incubated for 72 hours. Data shown

are representative of 3 different experiments. Viable cells were detected by measuring

absorbance at 540 nm using a Syngery HT microplate reader (BioTek).

Matrigel Invasion Assay: BD Biocoat invasion chambers (8 µm pore size, BD

Biosciences) were used per the manufacturer’s instructions with 1x106 cells in serum free

media placed into the upper chamber. Media with 5% fetal calf serum was used as the

chemoattractant in the lower chamber. After incubation for 24 hours at 37°C, cells in

the upper chamber were removed with cotton swab. Cells on the undersurface of the

filter were processed and counted in a blinded fashion as for the Transwell assay.

CXCR3 Assays: Because MOC2 was derived from a CXCR3-/- mouse, all cell lines were

analyzed by FACS for cell surface CXCR3 and none expressed CXCR3 (data not

shown). Despite this, to further confirm that CXCR3 absence did not play a role in the

growth phenotype of MOC2, we enforced expression of CXCR3 into MOC2

2

RV-mCXCR3-GFP was created by inserting mouse CXCR3 into GFP-RV (Ranganath et

al., J Immunol. (1998) 161:3822-6.). Constructs were then retrovirally transduced into

MOC2 tumor cells.

RAS sequencing: All three ras isoforms and the relevant B-Raf regions were sequenced

from tumor cell line cDNA as described by Dam et al. (BMC Cancer. 2006;6:177).

RAS activation assays: Activated RAS was detected using the Ras Activation Assay Kit

(Cell Biolabs). Briefly, total cell lysates were incubated with Raf1 RBD agarose beads to

pull down activated GTP bound RAS. Lysates were also independently treated with GDP

(negative) or GTPγS (positive) for controls. The Raf1 RBD bound protein was then

analyzed by Western blot and probed with antibody to either total RAS (Cell Signaling)

or K-RAS (Santa Cruz Biotechnology).

FACS Analysis: Scramble or shRNA transduced mouse tumors were harvested at the end

of tumor growth experiments and single cell suspensions were generated by chopping and

collagenase IA (Sigma-Aldrich) digestion. Cells were blocked with Fc Block

(Biolegend) and stained with CD45 FITC (Biolegend), CD44 PE (Biolegend) and 7-AAD

(BD Biosciences). A FACSCalibur was used to collect 10,000 CD45-/7-AAD- events and

data were analyzed with FloJo (Tree Star). Human tumors were collected under an IRB

approved protocol at Stanford University. Tumors were processed with modifications of

the protocol of Boiko et al. (Nature, 466:133-7). Briefly, tumors were minced and

digested overnight in 300 U/mL collagenase and 100 U/mL hyaluronidase (STEMCELL

3

Technologies). This suspension was then pelleted and briefly trypsinized (3 minutes) and

then treated with a Dispase/DNAse I solution (Roche). Cell were filtered, treated with

Ack RBC lysis buffer (Lonza), washed and used for staining. Single cell suspensions

were then blocked and surface stained with CD45-FITC and CD31-FITC. Phospho-flow

was performed as decribed by Nolan and colleagues using p-ERK1/2-PE and CD44-APC

(Zampieri et al. (2007) J of Virology, 81:1230-1240). We used the “fluorescence minus

one (FMO)” approach as described by Herzenberg et al. (Nature Immunology (2006),

7:681-685) to set thresholds and events were collected and data analyzed using FloJo

(Tree Star).

Supplementary Figures

Figure S1: Primary MOC Tumors and corresponding histology. A. Primary oral

cavity parental tumors for MOC1, 2, 7. B. H&E histology of primary parental tumors

showing moderately differentiated squamous cell carcinomas (40X magnification). C.

Immunofluorescence of MOC cell lines and control fibrosarcoma (H31m1) showing that

MOC cell lines have an epithelial phenotype with positive cytokeratin staining (green)

and H31m1 fibrosarcoma has negative cytokeratin staining (green) (40X magnification).

All cell lines have nuclear staining with DAPI.

Figure S2: Flank injection of MOC tumor cell lines, orthotopic injection of MOC2

and lung metastases of MOC10 A. Tumor growth curves of indicated 5 MOC cell lines

with each line on graph representing an individual mouse (n=4-5 mice per cell line).

Note that MOC22 demonstrated a cystic growth pattern thus gross tumor volume is likely

4

overestimated by external measurements (indicated by asterisk). B. MOC2 (30,000

cells) were injected in a 30 µl volume into the right buccal/floor of mouth region and a

representative image of a day 14 mouse shows rapid, locally invasive growth (black

arrow). C. Representative image of cervical metastases (black arrow) with primary

tumor in buccal/floor of mouth region (white arrow). D. MOC10 lung metastases from

flank injection of tumor cells (1X104)—multiple white nodules (black arrows) were seen

studding the lung surface and are shown magnified in the inset. E. H&E staining of

MOC10 lung metastases (20X magnification).

Figure S3: CXCR3 expression does not affect MOC2 growth in vivo. A. 1x105

MOC2 cells transduced with GFP or CXCR3-GFP were transplanted into the right flank

of C57BL/6 WT or C57BL/6 RAG2-/- mice and monitored for growth.

Figure S4: RAS mutation and activation status of MOC cell lines A. RAS isoforms

(data not shown for N-RAS) were sequenced and revealed that H-RAS mutations were

restricted to MOC1 and 22 and K-RAS mutations were present in MOC2, 7 and 10. B.

RAS activation assay with negative and positive controls (input protein was 1 mg of

lysate), manufacturer’s K-RAS control, total RAS (active and inactive) in 30 µg of lysate

from each cell line (10 µg for MOC23) and Raf1 RBD bound active RAS (pull down

from 2.5 mg of total protein for all cell lines except MOC23 which had 200 µg—

indicated by asterisk). Blot was probed with anti-total RAS (upper) and anti-K-RAS

(bottom). The K-RAS bands were quantitated using the Li-cor Odyssey (Li-Cor) and are

5

as indicated. Note that it is well established that mutant RAS/K-RAS bands have altered

migration in Western analysis (Srivastava et al., PNAS (1985) 82:38-42).

Figure S5: U0126 treatment decreases MOC cellular invasion but does not effect

proliferation. A. Western blot of p-ERK1/2 and ERK1/2 in MOC2 cells treated with

vehicle control (DMSO) or U0126 (10µM) for 24 hours. B. MTT proliferation assay of

MOC1 and MOC2 cells treated with increasing concentrations of U0126 (0, 0.1, 1, 10, or

20µM) for 24 hours. C. Representative 20x microscopic image of a Matrigel invasion

assay with MOC2 cells treated with vehicle control (DMSO) or U0126 (10µM). D.

Quantitation of Matrigel invasion assay where 4 random sections per filter x 3 filters

were counted in a blinded fashion by light microscopy at 20x magnification for both

MOC1 and MOC2 cells treated with DMSO or U0126 (***=p<0.001 for independent

samples t-test. A two way ANOVA showed that there is a significant difference between

vehicle and U0126 and this is different in MOC1 and MOC2 lines (significant interaction

effect -Line*vehicle F(1,28)=42.434, p<0.001)).

Figure S6: CD44 shRNA knockdowns inhibit in vitro migration and in vivo growth

but do not alter ERK1/2 activation. A. FACS analysis of cell surface CD44

expression in MOC2 after shRNA knockdown of CD44 with 3 distinct shRNAs (CD44-6,

CD44-7, CD44-10) or scramble control shRNA with the indicated MFI. B. Cell viability

of MOC 2 and 10 CD44 knockdown cells as assessed by MTT proliferation assay. C.

Western Blot of p-ERK1/2 and ERK1/2 for MOC2 cells after transduction with scramble,

CD44-6, CD44-7 or CD44-10 shRNAs (similar data not shown for MOC10). D. Scratch

6

Test of MOC2 after transduction with indicated CD44 or scramble shRNAs. E. MOC10

cells (1x104) transduced with scramble or CD44-6 shRNA were injected into the right

flank of C57BL/6 WT mice and monitored for growth. Growth curves only represent

average tumor diameter of transplanted tumors that grew out. F. MOC2 and MOC10

cells (1x105) transduced with scramble, CD44-6, or CD44-7 shRNA were injected into

the right flank of C57BL/6 WT mice and monitored for growth. For both E and F, a

mixed between-within subjects analysis of variance comparing scramble with the

indicated CD44 knockdown showed significant differences in tumor growth (*=p<0.05,

**=p<0.01, and ***=p<0.001).

Figure S7: In vivo escape of CD44 shRNA expressing tumors A. Gating strategy for

analysis of CD44 levels on transplanted tumors—single cell suspensions of flank

transplanted MOC10-scramble cells were first gated by forward and side scatter,

hematopoietic (CD45+) and dead (7-AAD+) cells were excluded and the remaining tumor

cells were analyzed for isotype control or CD44. B. Composite histograms of three

representative tumors from individual mice (of 2-3 mice per group) shows that both

MOC10-6 and MOC10-10 tumors had recovered CD44 to MOC10-scramble levels. For

starting CD44 levels, see Figure 3C

Figure S8: Immunofluorescence staining of transplanted MOC1 and MOC2 lines

for CD44 and p-ERK1/2 Individual panels of merged image shown in Figure 4A (all

images are at 20X). Scale bar in MOC1 DAPI stain represents 100 µM—the same scale

7

applies to all images. Individual panels between MOC1 and MOC2 were all captured at

the same microscope settings.

Figure S9: U0126 treatment decreases CD44 expression on MOC2, 7, 10 and UPCI:

SCC029B and tamoxifen does not induce CD44 on MOC1. A. FACS analysis of cell

surface CD44 expression after MOC2 cells were treated with vehicle control (DMSO,

gray line) or U0126 (10µM, black line) for 24 hours with the indicated MFI. B. FACS

analysis of cell surface CD44 expression after MOC7 and MOC10 cells were treated with

vehicle control (DMSO, gray line) or U0126 (10µM, black line) for 48 hours with the

indicated MFI. C. FACS analysis of cell surface CD44 expression after MOC1 cells

were treated with (black line) or without (gray line) tamoxifen for 48 hours. Isotype

control is represented by the gray shaded curve and MFIs are as indicated. D. FACS

analysis of cell surface CD44 expression after human OSCC cell line UPCI: SCC029B

was treated with vehicle control (DMSO, gray line) or U0126 (10µM, black line) for 48

hours with the indicated MFI.

Figure S10: Gating strategy for freshly resected human oral carcinomas. From left

to right are shown the specific gates used to analyze p-ERK1/2 staining in CD44high

versus CD44low tumor cells for the 7131 primary human tumor. The fluorescence minus

zero (FMO) and p–ERK1/2 stains are shown. The left three panels utilized forward and

side scatter gates to select the initial populations. Subsequently, hematopoietic and

endothelial markers were used to select tumor cell populations which were finally

8

analyzed for CD44 and p-ERK1/2. The CD45-/CD31- gating and dot plots of human

tumors 7132 and 7133, the latter is also in main figures, are also shown below.

Judd et al., Figure S1

A

B

MOC1 MOC2 MOC7

A

B

MOC1 MOC2 MOC7

OSCC10

C

H31m1

0 20 40 60

0

5

10

15

20

25

0 20 40 60

0

5

10

15

20

25

0 20 40 60

0

5

10

15

20

25

0 20 40 60

0

5

10

15

20

25

0 20 40 60

0

5

10

15

20

25

Avg

Tum

or D

iam

eter

(mm

)

MOC1 MOC2 MOC7 MOC10 MOC22* A.

SCC#

B. MOC2 FOM

D. MOC10 Lung

C. Neck LN Met


E. Lung H&E

DAY

A

0 10 20 30

0

5

10

15

20WT CXCR3 GFP

WT GFP

RAG2-/- CXCR3 GFP

RAG2-/- GFP

DAY

Ave

rage

Tum

or D

iam

eter

(mm

)


0 10 20 30

0

5

10

15

20WT CXCR3 GFP

WT GFP

RAG2-/- CXCR3 GFP

RAG2-/- GFP

DAY

Ave

rage

Tum

or D

iam

eter

(mm

)

Blot: pan Ras

Blot: Kras

Total protein (30 µg/lane)

Ac?vated Ras Raf1 RBD bound (2.5 mg/lane)

Cell Line Hras Kras BRaf

MOC1 Q61L -‐ -‐

MOC2 -‐ Q61H -‐

MOC7 -‐ Q61H -‐

MOC10 -‐ Q61H -‐

MOC22 Q61L -‐ n.d.

MOC23 -‐ -‐ n.d.

A. B.


*All mutations are AàT transversions

Activated K-ras levels

MOC1MOC2

MOC7

MOC10

MOC220

5

10

15

20

25

Ban

d In

tens

ity

vehicl

e

0.1uM U

0126

1uM U

0126

10uM U

0126

20uM U

0126

0.0

0.2

0.4

0.6

0.8

Ave

rage

Abs

orba

nce

at 5

40nm

vehicl

e

0.1uM U

0126

1uM U

0126

10uM U

0126

20uM U

0126

0.0

0.1

0.2

0.3

0.4

Ave

rage

Abs

orba

nce

at 5

40nm

MOC2 veh

icle

MOC2 U01

26

MOC1 veh

icle

MOC1 U01

260

50

100

150

Ave

rag

e N

um

be

r o

f C

ells P

er

20

x F

ield

A

MOC1

MOC2

B

MOC2

vehicle U0126

C

D

***

***

MOC2 p-‐ERK1/2 ERK1/2

vehicle

U0126


0 20 40 60

0

5

10

15

20

25

DAY

Ave

rag

e T

um

or D

iam

ete

r (m

m)

0 20 40 60

0

5

10

15

20

25

DAY

Ave

rag

e T

um

or D

iam

ete

r (m

m)

0 15 30 45 60

0

5

10

15

20

25

DAY

Ave

rag

e T

um

or D

iam

ete

r (m

m)

0Hours 24Hours

MOC2Scramble CD44‐6 CD44‐7 CD44‐10

100

101

102

103

104

CD44-PE

0

20

40

60

80

100

% o

f Max

A Isotype

Scramble(MFI=64.3)

CD44‐6(MFI=24.8)

CD44‐7(MFI=4.8)

CD44‐10(MFI=45.4)

Isotype Control Scramble (MFI=64) CD44-6 (MFI=25) CD44-7 (MFI=5) CD44-10 (MFI=45)

MOC2

C D

0 20 40 60

0

5

10

15

20

25

DAY

Ave

rag

e T

um

or D

iam

ete

r (m

m)

MOC10 MOC2

0 20 40 60

0

5

10

15

20

25

CD44-7

Scramble

DAY

Ave

rag

e T

um

or D

iam

ete

r (m

m)

0 15 30 45 60

0

5

10

15

20

25

CD44-6

Scramble

DAY

Ave

rag

e T

um

or D

iam

ete

r (m

m)

F

**

***

0 20 40 60

0

5

10

15

20

25

DAY

Ave

rag

e T

um

or D

iam

ete

r (m

m)

**

*

E MOC10

3/5 **

5/5

ERK1/2

p-‐ERK1/2

MOC2

100

101

102

103

104

CD44-PE

0

20

40

60

80

100

% o

f Max


0.0

0.1

0.2

0.3

0.4B MOC2 MOC10

0.0

0.2

0.4

0.6

A54

0 Scramble CD44-6 CD44-7 CD44-10

A. Ga?ng strategy (shown for MOC10-‐scramble)

B. Composite representa?ve data of in vivo CD44 shRNA loss


Forward Scatter

Side

Sca

tter

76.5

CD45 FITC7-

AA

D

48.4

Isotype PE

CD44 PE

Isotype

MOC10 Scramble

MOC10-6

MOC10-10

MOC1

MOC2

DAPI CD44 P-‐ERK1/2 MERGE


MOC2-24 Hours MOC7 MOC10 A B

C D

Isotype Control MOC1 (MFI=11.6) MOC1+T (MFI=11.1)

100

101

102

103

104

CD44-PE

0

20

40

60

80

100

% o

f Max

UPCI: SCC029B MOC1

CD44-PE CD44-PE CD44-PE

CD44-PE10

010

110

210

310

4

CD44-PE

0

20

40

60

80

100

% o

f Max

vehicle (MFI=121) U0126 (MFI=83)





FSC-A

SSC-

A

79.6

FSC-H

FSC-

W

78.4

SSC-H

SSC-

W

94.5

<FITC-A>: CD45/CD31

# Ce

lls

45.5

<PE-A>: pERK12

<APC

-A>:

CD

44

26.2 23.1

4.4846.2

FMO pERK1/2 Exclusion of doublets in FSC and SSC Exclusion of CD45+/CD31+

Ga?ng Strategy for p-‐ERK1/2 staining on primary human oral cancers


7131

7132

7133

CD44

supplementary methods, figures 1-10

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