32

SUPPLEMENTARY MATERIALS

Supplementary Methods

Supplementary Figure 1: Surface Expression Of ROR1 Associates With Breast Cancer

Cell-lines That Have High-metastatic Potential.

Supplementary Figure 2: High-level Expression of ROR1 In Breast Cancer Is Associated With

Shorter Lung, Bone and Brain Metastasis-free Survival.

Supplementary Figure 3: High-Level Expression Of ROR1 In Breast Cancer Is Associated With

Shorter Metastasis-Free Survival, and Independent from their ER, PR and HER2 status. Supplementary Figure 4. Expression of ROR1 By Breast Cancer Cell Lines Is Associated With

Features Of EMT.

Supplementary Figure 5: Silencing ROR1 Reduces Expression Of CXCR4.

Supplementary Figure 6. Silencing ROR1 Regulates EMT Genes expression.

Supplementary Figure 7: Silencing ROR1 Effects Modest Late-Growth Inhibition Of Orthotopic

Xenografts At The Site Of Injection But Strong Inhibition Of Experimental Pulmonary

Metastases.

Supplementary Figure 8: Immunohistochemistry of Experimental Metastatic Foci.

Supplementary Figure 9. Silencing ROR1 Reduces Pulmonary Metastasis And Bone

Metastasis Of MDA-MB-231 Derived Cell lines LM2-4175 And BoM-1833 In Vivo.

Supplementary Figure 10: Silencing ROR1 Inhibits Migration Of HS-578T and BT549 Migration

In Vitro.

Supplementary Table 1: Summary Of Human Breast Cancer Cell Line Characteristics With

Respect To EMT Status, Subgrouping, Invasiveness And Matrigel Morphology

Supplementary Table 2: Correlation Of ROR1 Status With Various Clinical Features Of Breast

Cancer Supplementary Table 3: Expression Of ROR1 Serves As An Independent Risk Factor For Short

Metastasis-free Survival

33

Supplementary Methods

Silencing of human ROR1

Silencing ROR1 was achieved by targeting the sequences 5′-TCCGGATTGGAATTCCCATG-3′

(shRNA1), and 5′- CTTTACTAGGAGACGCCAATA-3′ (shRNA2) as previously described (1).

The viral particles used to transduce breast cancer cell-lines were obtained by transfection of the

293-FT packaging cell line, and collected from cell supernatants at 48 and 72 h

post-transfection. Supernatants were filtered and centrifuged at 43,000xg to concentrate the

virus particles, which were used to infect sub-confluent cultures in the presence of

5 μg/ml polybrene overnight. Twenty-four hours post-transfection, cells were selected in media

containing 2 μg/ml puromycin. Cells silenced for ROR1 were sorted by flow cytometry using an

anti-ROR1 mAb (Clone 4A5). Sorted cells stably expressing shRNA1 or shRNA2 were

designated ROR1-shRNA or ROR1-shRNA2, respectively. Cells were additionally transduced

with lentivirus luciferase-GFP. Labeled cells were pooled and sorted for expression of GFP via

flow cytometry. Pooled populations of ROR1-silenced cells and luciferase-GFP labeled cells,

obtained in the first 10 generations after cell sorting without subcloning, were injected into

RAG-/-γc-/- mice for the in vivo experiments. The efficiency of the ROR1 silencing was confirmed

by SYBR green qRT-PCR (Applied Biosystems, Carlsbad, CA), or immunoblot analysis with an

anti-ROR1 antibody (Cell Signaling, Danvers, MA). Expression of GAPDH and β-actin were

used as endogenous controls for qRT–PCR and immunoblot analysis, respectively. ROR1

siRNA (s9755) or non-targeting (control) were purchased from Life TechnologiesTM. All siRNA

transfections were performed in DMEM serum-free medium using lipofectaimine RNAiMAX

(Invitrogen) according to the manufacturer's instruction.

34

Transfection Of ROR1-negative Cells With Vectors Encoding Human ROR1

MCF7-CTRL, MCF7-ROR1 stable transfectants were generated as previously described (1) .

The human ROR1 cDNA was cloned into the pcDNA3 vector and then transfected into MCF7 cell

line, using the Amaxa electroporation nucleofector™. Stable cell lines were sorted by flow

cytometry using anti-ROR1 antibody (Clone:4A5, conjugated to Alexa Fluor-647) and cultured in

DMEM with 1.5 mg ml-1 G418.

Trans-well migration and invasion assays

Cancer cells were cultured overnight in Dulbecco’s modified Eagle’s medium (DMEM)

supplemented with 0.2% fetal bovine serum (FBS) without growth factors. The following day,

cells were removed from the plastic via treatment with trypsin and suspended in 0.2% FBS

DMEM media without growth factors. Tumors cells were seeded at a density of 25,000 cells per

well into trans-well inserts (3 μM pore size, BD Biosciences, San Jose, CA) for migration assays,

or at a density of 50,000 cells per well into Matrigel-coated, growth-factor-reduced, invasion

chambers (8 μM pore size, BD Biosciences). Migration assays were performed using tumors

cells pre-treated with control non-specific mouse IgG (Invitrogen, Carlsbad, CA) or D10 at 40

μg/ml for 1 hour at 37o C. The lower chambers were filled with culture medium containing 5%

FBS or 200 ng/ml CXCL12, as chemoattractant. Wells were washed with phosphate buffered

saline (PBS) and fixed with 4% paraformaldehyde after 6 h for migration assays or after 22 h for

invasion assays. The cells on the apical side of each insert were removed by scraping. Cells

that had migrated to the basal side of the membrane were stained by Diff-Quick staining kits

(IMEB Inc., San Marcos, CA) and visualized with a Nikon inverted microscope.

35

Analysis of mRNA and protein expression

Total RNA was purified using the Qiagen RNeasy kit (Valencia, CA). Quantitative reverse

transcriptase polymerase chain reaction (qRT-PCR) was performed as previously described (1).

Protein expression levels were assessed via immunoblot analysis with cell lysates (40–60 μg)

prepared in lysis buffer (20 mM HEPES (pH 7.9), 25% glycerol, 0.5 N NaCl, 1 mM EDTA, 1%

NP-40, 0.5 mM dithiothreitol, and 0.1% deoxycholate) containing protease inhibitors (Roche, SF,

CA) using anti-SNAIL-1, anti-SNAIL-2, anti-ZEB1, anti-ZO-1, anti-vimentin, anti-ROR1, or

anti-β-actin antibodies from Cell Signaling, and anti-CK-19 from EMD Millipore (Billerica, MA).

For immunoprecipitation studies, cells were lysed in buffer containing 1% NP40, 10mM Tris-HCl

(pH7.5), 150mM NaCl, and 1mM EDTA with protease inhibitors (Roche). Cell lysates were

incubated with indicated antibodies for 3 h at 4° C. We then added 10μl 50% protein-A

Sepharose beads and incubated the mixture with gentle agitation for 1 h at 4° C. Bound beads

were washed four times in lysis buffer and incubated in SDS-sample buffer to elute bound protein,

which was examined via immunoblot analysis.

Flow cytometry

Breast cancer cells were sorted using BD FACSAriaTM flow cytometry (BD Biosciences). Cells

were washed and suspended in 2% bovine serum albumin (BSA) (Sigma, St. Louis, MO) in PBS

and stained for ROR1 using an Alex488-conjugated antibody (clone 4A5 or clone D10) or an

Alex488-conjugated IgG2b or IgG2a isotype control. Flow cytometry data were collected using

a FACSCaliburTM cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star

Inc, Ashland, OR). Antibody-mediated ROR1 internalization was performed by flow cytometry,

as previously described (2).

36

Immunofluorescence and immunohistochemistry analysis

Mouse lungs were fixed with 4% paraformaldehyde and embedded in paraffin or snap-frozen in

Optimal Cutting Temperature (OCT) compound for subsequent histological examination.

Five-μm-thick tissue sections were prepared and stained with Hematoxylin & Eosin (H&E) or

anti-phospho-AKT (Ser473, D9E, Cell Signaling), anti-phospho-CREB (Ser133, 87G3, Cell

Signaling), anti-CK-19 (RCK108, Dako, Carpinteria, CA), or anti-vimentin (D21H3, Cell Signaling)

primary antibodies. Images were collected using a Delta Vision microscope and processed with

SPOT software. For internalization immunofluorescence analysis, cells were stained with the

D10 mAb, followed with a goat anti-mouse Ig-Alex488 (Invitrogen) on ice. The stained cells

were kept on ice or transferred to 37o C for 1 h. Cells were then fixed with 4%

paraformaldehyde, mounted on slides undercover slips with DAPI mounting media (Sigma) and

sealed with clear nail polish. For immunofluorescence analysis, cells fixed with 4%

paraformaldehyde, permeabnlizized in 0.1% Trion X-100, blocked with 5% BSA, stained with

primary antibody anti-E-Caherein (1:200, Cell Signaling, #3195s), anti-Vimentin(1:100, Cell

Signaling, #5741s), or anti-CK-19 (1:200, Abcam, ab9221) followed by Alexa Fluro 488 and/or

Alexa Fluro594 secondary antibodies (Invitrogen). The cell nucleus were stained with DAPI.

Fluorescence images were obtained using laser scanning confocal imaging system (Olympus

FV1000).

Gene expression analysis from GEO datasets

We compiled 4 microarray datasets of 582 patients from the PubMed GEO database (Gene

Expression Omnibus database, http://www.ncbi.nlm.nih.gov/gds), as previously described (3) .

GSE2603, GSE5327, GSE2034, and GSE12276 datasets were transformed by log2 and each

microarray was centered to the median of all probes. For each patient, metastasis-free survival

37

was defined as the time interval between surgery and the diagnosis of metastasis. Patients

were subsequently sub-grouped in tertiles based on their relative expression of ROR1.

Analysis of metastasis

Experiments with RAG-/-γc-/- mice were carried out in accordance with the guidelines of the

National Institutes of Health on animal welfare. The UCSD Animal Care Program approved all

animal protocols (S03037). Female RAG-/-γc-/- mice were injected with the following:

ROR1-shRNA-transfected MDA-MB-231 (group 1), CTRL-shRNA-transfected MDA-MB-231

(group 2), ROR1-shRNA2-transduced LM2-4175 (group 3), CTRL-shRNA2-transduced

LM2-4175 (group 4), ROR1-shRNA2-transduced BoM-1833 (group 5), or

CTRL-shRNA2-transduced BoM-1833 (group 6). Cells were injected intravenously (i.v.)

through the lateral tail vein in 100 μl PBS (5 × 105 for groups 1-2; 2 × 105 for groups 3-4) or

administered by intracardiac (i.c.) injection in 100 µl PBS (1 × 105 for groups 1-2; 1 × 105 for

groups 5-6). Non-invasive bioluminescence imaging was performed weekly using an IVIS 200

imaging system (Caliper life sciences, Hopkinton, MA). All mice were euthanized at 3-4 weeks

post-injection, and their lungs were removed, weighted and fixed in 10% formalin. The

lung-weight-index is the ratio of lung weight to body weight.

To study the effect of ROR1 on the in vivo metastasis of mammary fat pad xenografts, we

injected 1x106 cells under second mammary fat pad area of the right abdominal mammary gland.

Tumor cells were suspended in 100 μl of phosphate buffered saline/Matrigel mixture (1:1

volume). We measured the tumor size every 3 days, and then surgically excised the tumors

when the tumor volume reached 300mm3. Non-invasive bioluminescence imaging was

performed on the day after implanting the orthotopic xenograft, the day of primary tumor removal,

38

and weekly for the following 3-week period. Mice were sacrificed 21 days after the primary

tumor was surgically removed, and the lungs, liver, and bone were removed and analyzed or

fixed in 10% formalin for histological evaluation.

To study the therapeutic effect of anti-ROR1 monoclonal antibodies in breast cancer metastasis,

5x105 MDA-MB-231 cells were injected via lateral tail vein of eight-week-old female RAG-/-γc-/-

mice. Control mouse IgG (5mg/kg) or anti-ROR1 mAb D10 (5mg/kg) was administered via

intravenous (i.v.) injection on days 1, 3, 7, 14, and 21 following tumor injection. Non-invasive

bioluminescence imaging was performed weekly. Three days or 5 weeks after establishment of

the xenograft, mice were sacrificed and the lungs were removed and fixed in 10% formalin for

histologic evaluation.

We monitored the growth of primary mammary fat pad xenografts or metastatic growth in the

lung or bone via bioluminescence imaging. For this we injected D-luciferin (150 mg/kg) into the

peritoneum of mice that had received i.v. or i.c. injections of tumor cells transfected to express

luciferase. Ten minutes later the anesthetized mice were imaged with Xenogen IVIS-200

machine (Caliper Life Sciences, CA). Bioluminescence analysis was carried out using Living

Image software 3.2 (Caliper Life Sciences). Bioluminescence photon fluxes, which were

proportional to the number of light emitting tumor cells, were normalized by day 1 for mammary

fat pad xenografts, or day 0 for lung or bone metastases.

Statistical analyses

The lung metastasis assay using MDA-MB-231 was repeated four times (n=5-8 for each cohort).

39

Results are shown as Figure 4B. The same result was reproduced independently in three

additional experiments. Bone metastasis assay using MDA-MB-231 cells was repeated twice (n

= 5-8 for each cohort; Fig. 4I-L). For the orthotopic xenograft (Fig. 3), lung metastasis survival

using MDA-MB-231 (Fig. 4A), lung colonization assay (Fig. 4C), bone metastasis survival using

MDA-MB-231 (Fig. 4H), anti-ROR1 antibody D10 treatment (Fig. 5), lung metastasis assay using

LM2-4175 (Supplementary Fig. S9D, S9F-H), lung metastasis survival using LM2-4175

(Supplementary Fig. S9E) and bone metastasis assay using BoM-1833 (Supplementary Fig.

S9I-K), each experiment was performed with n = 5-12 mice in each cohort. Metastasis-free

survival (MFS) was compared using Kaplan-Meier survival analysis. To test the differences

among MFS curves, the log rank test was used. Data are presented as means ± standard error

of the mean (SEM). An unpaired two-sided Student's t test was used to compare two groups

unless otherwise indicated. A p<0.05 was considered statistically significant.

REFERENCES

1. Zhang S, Chen L, Cui B, Chuang HY, Yu J, Wang-Rodriguez J, et al. ROR1 is expressed in

human breast cancer and associated with enhanced tumor-cell growth. PloS one.

2012;7:e31127.

2. Baskar S, Kwong KY, Hofer T, Levy JM, Kennedy MG, Lee E, et al. Unique cell surface

expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia.

Clinical cancer research : an official journal of the American Association for Cancer Research.

2008;14:396-404.

3. Zhang XH, Wang Q, Gerald W, Hudis CA, Norton L, Smid M, et al. Latent bone metastasis in

breast cancer tied to Src-dependent survival signals. Cancer Cell. 2009;16:67-78.

40

Supplementary Figure 1. Surface Expression Of ROR1 Associates With Breast Cancer

Cell-lines That Have High-metastatic Potential. Expression of surface ROR1 on various

41

breast cancer cell lines, as assessed via flow cytometry. Representative fluorescence

histograms of cells stained with anti-ROR1 mAb (ROR1, open histograms) or isotype-control

IgG (ISO, shaded histograms) are presented for the cell line indicated at the top of each

histogram. Mean fluorescence intensity (MFI) of the ISO- or anti-ROR1 stained cells is

indicated on the top-right corner of each histogram. (A) ROR1 expression in basal-type

breast-cancer cell-lines MDA-MB-231, HS-578T, or BT549, (top row) or MDA-MB-435S,

MDA-MB-436, or MDA-MB-157 (bottom row). (B) ROR1 expression in luminal-type,

breast-cancer cell-lines MCF7, BT474, MDA-MB-453, SKBR3, MDA-MB-330 or BT-483.

42

Supplementary Figure 2. High-level Expression of ROR1 In Breast Cancer Is Associated

With Shorter Lung, Bone and Brain Metastasis-free Survival. The graph was derived from

published data available through the PubMed GEO database (GSE2603, GSE5327,

GSE2034, and GSE12276). Kaplan-Meier curves depict the prognostic impact of ROR1

expression on (A) lung metastasis-free survival, (B) bone metastasis-free survival, or (C)

brain metastasis-free survival. For each analysis, 582 cases were segregated into tertiles

Lung relapse by ROR1 status

0 20 40 60 80 100 120 140 160 180 200

40

60

80

100

ROR1H 68-100thROR1M 34-67thROR1L 0-33rd

ROR1L

nRelapse status

NoYes

194194194

39ROR1MROR1H

2522

155

169172

p =0.03

p=0.002

Metastasis-free survival (months)

% w

/o L

ung

Rel

apse

Bone relapse by ROR1 status

0 20 40 60 80 100 120 140 160 180 200

40

60

80

100

ROR1H 68-100thROR1M 34-67thROR1L 0-33rd

ROR1L

nRelapse status

NoYes

180175169

117ROR1MROR1H

127124

63

4251

p=0.04

p=0.004

Metastasis-free survival (months)

% w

/o B

one

Rel

apse

Brain relapse by ROR1 status

0 20 40 60 80 100 120 140 160 180 200

40

60

80

100

ROR1H 68-100thROR1M 34-67thROR1L 0-33rd

ROR1L

nRelapse status

NoYes

172183169

158ROR1MROR1H

162174

14

79

p =0.24

p=0.04

Metastasis-free survival (months)

% w

/o B

rain

Rel

apse

A

B

C

43

with group designated ROR1H representing the one-third of the patients who had tumors with

the highest levels of ROR1 mRNA, and the group designated ROR1L representing the

one-third of patients who had cancers with the lowest levels of ROR1 mRNA. The one-third

of patients who had tumors with intermediate expression of ROR1 mRNA was designated as

ROR1M. Metastasis-free survival was determined by Kaplan-Meier analyses, and statistical

differences were determined by log-rank test. The number of patients in each category, the

total metastatic events, and the corresponding P values (chi-square test) are shown in the

embedded tables.

44

45

Supplementary Figure 3. High-level Expression of ROR1 In Breast Cancer Is Associated

With Shorter Metastasis-free Survival, and Independent from their ER, PR and HER2 status.

Cohort of 582 patients with breast adenocarcinoma included in survival analysis. (A)

Comparison of the levels of ROR1 mRNA expression of the malignant cells of ERNeg (n = 242)

and ER+ (n = 325) breast cancer patients (left panel), PRNeg (n = 274) and PR+ (n = 271)

breast cancer patients (center panel), and HER2Neg (n = 404) and HER2+ (n = 106) breast

cancer patients (right panel). Results are means ± SEM The p value was determined by

Student’s t-test. (B) Prognostic impact of ER status on overall-metastasis-free survival (P =

0.13 by log-rank test). (C) Prognostic impact of ER status and ROR1 mRNA expression on

overall-metastasis-free survival (P < 0.0001 by log-rank test). (D) PR status on

overall-metastasis-free survival (P = 0.0007 by log-rank test). (E) Prognostic impact of PR

status and ROR1 mRNA expression on overall metastasis-free survival (P < 0.0001 by

log-rank test). (F) HER2 status on overall-metastasis-free survival (P = 0.16 by log-rank test).

(G) Prognostic impact of HER2 status and ROR1 mRNA expression on overall

metastasis-free survival (P < 0.0001 by log-rank test).

46

Supplementary Figure 4. Expression Of ROR1 By Breast Cancer Cell Lines Is Associated

With Features Of EMT. (A) Immunoblots of lysates from MDA-MB-231 transfected with

CTRL-shRNA or ROR1-shRNA were probed with antibodies specific for ROR1 (top) or β-actin

(bottom) as indicated on the left. (B) Mean amount of VIM and KRT19 (± SEM), as detected

via qRT-PCR on triplicate samples. Data are shown as means ± SEM; *P < 0.05, **P < 0.01,

compared with CTRL-shRNA group.

47

Supplementary Figure 5. Silencing ROR1 Reduces Expression Of CXCR4. (A)

Histograms indicating the amount of CXCR4 mRNA detected via qRT-PCR in triplicate

samples of MDA-MB-231 transfected with either CTRL-shRNA2 or ROR1-shRNA2, as

indicated at the bottom of each histogram. (B) Representative flow cytometry fluorescence

histograms of ROR1-shRNA2 (open histogram with green line) or CTRL-shRNA2 (open

histogram with blue line) transduced MDA-MB-231 cells stained with anti-CXCR4-APC mAb

or isotype-control mAb (shaded histograms), respectively. (C) Cells were seeded into the

top chambers of trans-wells without BD MatrigelTM to examine for chemotaxis to CXCL12,

which added to a final concentration of 200ng/ml to the bottom chambers. The cells that

migrated after six-hours at 37o C were enumerated under 10x magnification. The histograms

each provides the numbers of migrated cells in each of three chambers seeded with

MDA-MB-231 cells transfected either with CNTL-shRNA or ROR1-shRNA, as indicated at the

bottom of the histogram. Results are representative of 3 independent experiments. Data

are shown as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, compared with

CTRL-shRNA group.

48

Supplementary Figure 6. Silencing ROR1 Regulates EMT Genes expression. Histograms

indicating the relative mRNA amount of variety genes, as indicated at the bottom of each

histogram, detected via qRT-PCR in triplicate samples of MDA-MB-231(A) , HS578T(B), and

BT549(C) transfected with either CTRL-siRNA or ROR1-siRNA. Results are representative

of 2 independent experiments. Data are shown as means ± SEM; *P < 0.05, **P < 0.01,

compared with CTRL-siRNA group.

49

Supplementary Figure 7. Silencing ROR1 Effects Modest Late-Growth Inhibition Of

Orthotopic Xenografts At The Site Of Injection But Strong Inhibition Of Experimental

Pulmonary Metastases. (A) RAG-/-γc-/- mice were given subcutaneous (s.c.) or intravenous

(i.v.) injections of CTRL-shRNA-transfected or ROR1-shRNA-transfected MDA-MB-231.

The bioluminescence photon flux of the primary tumor in the injected mammary fat pad or of

the lung of each mouse was normalized against the photon flux detected for the first

measurement following the injection of tumor (100 represents 100% of the photon flux

detected on the day of the initial measurement) (top panels). The top three graphs depict the

normalized bioluminescence photo flux of the mammary fat pads of mice given s.c. injections

50

of 1×106 (left), 5×105 (center), or 2.5×105 (right) indicated cells. The bottom graphs provide

normalized bioluminescence photo flux of the lung of mice given i.v. injections of 1×106 (left),

5×105 (center), or 2.5×105 (right) indicated cells. (note: the bottom left graph depicts the

actual mean bioluminescence photon flux of the lungs of mice given i.v. injections of 1×106

indicated cells. (B) The histograms depict the lung-weight-index for mice of each group on

d21 (n=5-8) i.v. injected with CTRL-shRNA-transfected (black) or ROR1-shRNA-transfected

MDA-MB-231 (grey) or no cells (white). The P values were determined by One-way ANOVA.

(C) H&E-stained sections of the lung representative of mice from each group on d21. Data are

shown as means ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, compared with CTRL-shRNA

group.

51

Supplementary Figure 8. Immunohistochemistry of Experimental Metastatic Foci.

RAG-/-γc-/- mice were given intravenous (i.v.) injections of 5x105 CTRL-shRNA-transfected

MDA-MB-231 (top panels) or ROR1-shRNA-transfected MDA-MB-231 (bottom panels). (A)

Sections of lung were prepared from animals euthanized on day 21. The lungs of mice

injected with ROR1-shRNA-transfected cells had few metastatic foci, which were identified for

immunohistochemistry analysis. The sections were stained with mAbs specific for Ki67+,

CK-19, or vimentin, or terminal deoxynucleotidyl transferase dUTP nick end labeling (Tunnel).

(40x magnification). (B) Sections of lung as in (a) were stained with mAb specific for

phospho-AKT (left panel) or phospho-CREB (right panel) (40x magnification).

52

Supplementary Figure 9. Silencing ROR1 Reduces Pulmonary Metastasis And Bone

Metastasis Of MDA-MB-231 Derived Cell lines LM2-4175 And BoM-1833 In Vivo. (A)

53

Schematic diagram showing that LM2-4175 cells preferentially metastasize to lung and

BoM-1833 cells preferentially metastasize to bone. Flow cytometry analyses showing the

ROR1 expression in LM2-4175 and BoM-1833. Mouse cartoons are modified from

reference (Cancer Cell, 2009;1;67-78) (B-C) Flow cytometry analyses showing the ROR1

silencing efficiency in LM2-4175 and BoM-1833, using ROR1-shRNA2. (D) Mice were each

given an i.v. injection of 2x105 CTRL-shRNA-transfected or ROR1-shRNA-transfected

LM2-4175 cells. Left, representative bioluminescence images of each group; Right,

normalized in vivo lung photon flux of each group. (E) Kaplan-Meier survival curves of mice

injected i.v. with 2x105 indicated LM2-4175 cells (P <0.0001 by log-rank test). (F) The

lung-weight-index of each group on d21 (bottom). Representative photos of the lungs of

each group (top). (G) The ex vivo lung GFP photon flux of each group on d21 (bottom).

Representative photos of the bones of each group (top). (H) Representative H&E-stained

histological sections of the lung on d21. (I) Mice were each given an i.c. injected of 1x105

CTRL-shRNA-transfected or ROR1-shRNA-transfected BoM-1833 cells. Top, representative

bioluminescence images of of each group; Bottom, normalized in vivo bone photon flux of

each group. (J) Representative bone ex vivo photon flux and H&E-stained histological

sections of the bone on d21. (K) Representative liver ex vivo photon flux and H&E-stained

histological sections of the liver on d21. Data are shown as means ± SEM; *P < 0.05, **P <

0.01, ***P < 0.001, compared with CTRL-shRNA group.

54

Supplementary Figure 10. Silencing ROR1 Inhibits Migration Of HS-578T and BT549 In vitro.

Data are shown as the means ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, compared with

cells treated with control IgG.

IgG D10 IgG D100

50

100

HS-578t BT549

** *

Rel

ativ

e M

igra

tion

(%)

55

Supplementary Table1. Summary Of Human Breast Cancer Cell Line Characteristics With Respect To EMT Status, Subgrouping, Invasiveness And Matrigel Morphology

Cell Line Matrigel Invasion ROR1 ΔMFI E-Cadherin N-Cadherin Vimentin Subtype ER PR HER2 MDA-MB-231 Stellate 4 High 60.2 0 N-Cad- VIM+ Basal - - -

HS-578T Stellate 4 High/Med 19.5 0 N-Cad+ VIM+ Basal - - -

BT549 Stellate 4 High 5.1 0 N-Cad+ VIM+ Basal - - - MDA-MB-435S Stellate 3 High 7.25 0 N-Cad+ VIM+ Basal - - - MDA-MB-436 Stellate 3 Med 3.45 0 N-Cad+ VIM+ Basal - - - MDA-MB-157 Stellate 3 Med 3.78 0 Basal - - - MDA-MB-134 Spherical 2 Low 5.44 0 VIM- Luminal + - - MCF7 Fused 2 Low 1.45 2 N-Cad- VIM- Luminal + + -

BT474 Fused 2 Low 1.01 2 VIM- Luminal + + +

MDA-MB-453 Spherical 2 Low 1.64 0 N-Cad- VIM- Luminal - - + SKBR3 Spherical 2 Low 0.74 0 N-Cad- VIM- Luminal - - + MDA-MB-330 Spherical 2 Low 0.1 Luminal - -  

BT-483 Fused 2 Low 0.3 2 VIM- Luminal + + - T47D Spherical 2 Low/Med 0.1 VIM- Luminal + normal

Matrigel morphology, Invasion stages and status of E-cadherin, N-Cadherin, Vimentin, subtype, ER, PR, HER2 are summarized

from previous studies listed below. Expression of surface ROR1 on various breast cancer cell lines was assessed via flow

cytometry. ROR1 expression shown as Delta Mean Fluorescence Intensity (ΔMFI) obtained by subtracting the isotype control

MFI from anti-ROR1 antibody staining MFI.

Studies summarized include (1) Michael et al. Cancer Research (1999) 59:947-952. (2) Monica MORINI, et al. Int. J. Cancer (2000)

87:336-342. (3) T. Blick et al. Clin Exp Metastasis (2008) 25:629–642

56

Supplementary Table2. Correlation Of ROR1 Status With Various Clinical Features Of

Breast Cancer

ROR1H ROR1M ROR1L p-value

ER ER+ 81 122 122 <0.0001 ERNeg 102 70 70 NA 11 2 2

PR PR+ 70 99 102 0.005 PRNeg 106 86 82 NA 22 5 10

HER2 HER2+ 23 42 41 0.045 HER2Neg 137 131 136 NA 34 21 17

T-Stage T1 61 75 64 0.18 T2 92 85 68 T3 10 7 9 T4 6 1 2 NA 26 25 51

Size (mm) 25.2 22.0 19.8 0.005 Age 50.6 52.3 54.2 0.02

The table was derived from published data available through the PubMed GEO database

(GSE2603, GSE5327, GSE2034, and GSE12276). 582 cases were segregated into tertiles

with ROR1H group , ROR1M group or ROR1L group based on ROR1 mRNA expression. For

analysis of estrogen receptor (ER), progesterone receptor (PR), HER2 and T-Stage by TNM

classification, the p-values were calculated based on Pearson Chi-Square Test. For tumor

size and age of patients, p-values were calculated based on One Way ANOVA test. NA =

not available.

57

Supplementary Table3. ROR1 Performs Independently Of Conventional Pathological

Factors In Metastasis-free Survival of Patients With Breast Cancer.

The table was derived from published data available through the PubMed GEO database

(GSE2603, GSE5327, GSE2034, and GSE12276). 582 cases were segregated into tertiles

with ROR1H (high) group , ROR1M (intermediate) group or ROR1L (low) group based on

ROR1 mRNA expression.

The performance of ROR1H in predicting metastasis-free survival was analyzed by

multivariate analyses with Cox proportional hazard regression models. The hazard ratio of

each covariate and its 95% confidence interval are reported. p-values were calculated

based on the Normal Distribution, assessing the probability for the null hypothesis (hazard

ratio = 1, i.e. no prognostic significance) to be true.

Overall Lung Bone Hazard Ratio p Hazard Ratio p Hazard Ratio p

ER 0.8 [0.6, 1.1] 0.13 0.3 [0.2, 0.5] < 0.0001 1.0 [0.7,1.4] 0.93 PR 0.6 [0.5, 0.8] < 0.0001 0.3 [0.2, 0.5] < 0.0001 0.7 [0.5, 1.0] 0.065

HER2 1.3 [0.9, 1.8] 0.16 0.9 [0.5, 1.6] 0.70 1.6 [1.0,2.4] < 0.05 ROR1H 2.3 [1.7, 3.0] < 0.0001 2.4 [1.5, 3.8] < 0.001 1.8 [1.3, 2.6] 0.001