www.sciencemag.org/cgi/content/full/science.1185837/DC1

Supporting Online Material for

Induction of Lymphoidlike Stroma and Immune Escape by Tumors that Express the Chemokine CCL21

Jacqueline D. Shields, Iraklis C. Kourtis, Alice A. Tomei, Joanna M. Roberts, Melody A. Swartz*

*To whom correspondence should be addressed. E-mail: melody.swartz@epfl.ch

Published 25 March 2010 on Science Express

DOI: 10.1126/science.1185837

This PDF file includes

Materials and Methods Figs. S1 to S7

Supporting online material

Materials and Methods

Tumor cell lines

B16-F10 melanoma cells (ATCC) derived from C57BL/6 mice were maintained in DMEM

supplemented with 10% FBS and penicillin-streptomycin-amphotericin B. shRNA knockdown

melanoma cells (CCL21low) were created using lentiviral transduction of shRNA for murine

CCL21, or scrambled shRNA (Mission shRNA, Sigma-Aldrich) and CCL21high cells by lentiviral

transduction of murine CCL21serine cDNA. Briefly, lentiviral vectors containing either

mCCL21 shRNA or scrambled shRNA were purchased from Sigma, while mCCL21ser cDNA

from pORF-mExodus2 v2.1 (Invivogen) was subcloned into the PRRLSIN.CPPT.PGK.WPRE

lentivector. Lentivectors carrying either mCCL21ser cDNA, mCCL21 shRNA or scrambled

shRNA were then transfected into 293T cells together with the pCMVR8.74 packaging plasmid

and pMD2.G envelope plasmid in a ratio of 3:2:1. Medium was collected after 48 hours and the

virus was concentrated by ultracentrifugation. B16-F10 cells were transduced with either the

lentivirus carrying mCCL21 shRNA (CCL21low) or scrambled shRNA (control), or mCCL21ser

cDNA (CCL21high) at a multiplicity of infection of 104. Stably transduced cells were selected

through antibiotic resistance to puromycin according to the manufacturers guidelines. Clones

were generated by serial dilutions and expanded; clones were chosen following ELISA and PCR

for mCCL21. Beta tumor cells (βTC) were transduced with lentivectors carrying mCCL21ser

cDNA in the same way to overexpress mCCL21.

13

Animals

6-8 week old female C57BL/6 mice (Charles River), 129S2 and 129P2 mice (Harlan), and

BALB/C mice (Charles River) were used for syngeneic and allograft models. CCR7-/- mice on a

C57CL/6 background (27) were a kind gift of Sanjiv Luther, the C57BL/6 Rorc(γt)+/GFP and

Rorc(γt)-GFPGFP/GFP (129P2/SJL backcrossed onto C57BL/6) mice (22) were a kind gift of

Gerard Eberl. For all in vivo experiments, 500,000 tumor cells were suspended in 50 μl sterile

saline and inoculated subcutaneously dorso-ventrally in the anesthetized mouse. All procedures

were carried out in accordance with Swiss law. For blocking studies, mice received 25 μg/mouse

i.p. every 2 days of either neutralizing anti-mouse CCR7 or matched control IgG (both from

eBioscience).

Cell isolation

Mice were anesthetized and sacrificed by cervical dislocation. Tumor volumes (V) were

estimated by the formula V = π/6 Lw2, where L = longest dimension and w = shortest dimension

of the tumor. Tumors and LNs (draining brachial and axillary LN, and non-draining inguinal LN)

were incubated in collagenase D (1 mg/ml in HBSS with 2% FBS) for 3h (tumors) or 30 min

(lymph nodes) at 37°C. The reaction was quenched with 100mM EDTA. Splenocytes were

isolated following mechanical disruption of spleens. Cell suspensions were passed through a 70

μm cell strainer and washed with HBSS.

Peritoneal macrophage isolation

Murine macrophages were isolated by injecting Brewers Thioglycollate medium i.p. into

C57BL/6 mice. After 4 days, mice were sacrificed and the peritoneal cavity wall was exposed.

14

10 ml sterile saline was injected into the peritoneal cavity and mixed, and the macrophage-rich

fluid was withdrawn. Cell suspensions were passed through 40 μm cell strainers and

characterized phenotypically by flow cytometry analysis before being assayed in vitro.

Antibodies and flow cytometry

The following anti-mouse antibodies were used for flow cytometry: CD45-APC, CD45-Pacific

blue, or biotinylated CD45, CD3ε-Pacific blue or CD3ε-PerCPCy5.5, CD4-PECy7 or CD4-PE,

CD8α-APC-Alexa 780, B220-PECy5, F4/80-PE, CD25-FITC, FoxP3-PerCPCy5.5, CD11c-

Alexa 647, MHCII-FITC, biotinylated Gr1, biotinylated Crry, gp38-Alexa 488, IL-7Rα-APC,

ROR-γt-PE, and CD11b-PECy7 (all from eBioscience). Pentamer staining for Trp2 H-2kb-PE

(ProImmune) was performed according to manufacturers guidelines. Antibodies were prepared

in 24G2 hybridoma medium and HBSS/0.2% BSA and added to samples prior to incubation at

4ºC for 30 min in the dark. Samples were washed and those requiring secondary antibody were

incubated for an additional 15 min with Streptavidin-conjugated pacific orange (Μolecular

Probes). Propidium iodide or Live/dead fixable labels (Μolecular Probes) were used to sort out

dead cells. Analysis was performed on a CyAn ADP Flow Cytometer (DAKO).  

Dendritic cell trafficking in vivo

B16-F10 CCL21low, control or CCL21high tumor cells were implanted as described. After 4 days,

tumors were inoculated with 20µl of 0.5 µm FITC-conjugated latex microspheres (Polysciences,

diluted 1:25 in sterile saline). 24 h later, tumors and lymph nodes (axillary and brachial) were

harvested and single cell suspensions were generated as described above. Cells were stained and

analyzed by flow cytometry.

15

ELISA

Tumor samples, snap frozen at the time of extraction, were homogenized in the presence of lysis

buffer of Tissue Protein Extraction Reagent (TPER, Pierce) and a protease inhibitor cocktail

(Complete Mini protease inhibitor cocktail tablets, Roche). Tumor homogenates were then

assessed for CCL21, TFG-ß1, IL-10, IL-2, CCL2, C5a, IL-4, IL-1ß and IFN-γ using ELISA kits

(R&D Systems, Preprotech, Cell Sciences and Ebioscience) according to manufacturer’s

instructions. Conditioned media from 2D cultures of normal human breast epithelial cells

(MCF10A), breast cancer (MCF7, ZR75-1, TD-47), prostate (DU145), osteosarcoma (U2OS)

and melanoma cells (MDA-MB-435, ME275-EP) were collected and analyzed for CCL21

expression by ELISA.

Immunofluorescence

Frozen samples of tumors, non-draining axillary lymph nodes, and normal skin were

cryosectioned (10μm) and subjected to standard immunofluorescence protocols using the

following anti-mouse antibodies: FITC-conjugated rat anti-CD31 (1:100, BD Pharmingen),

rabbit anti-LYVE-1 (1:500, RELIATech), goat anti-gp38 (1:75, R&D Systems), rat anti-ERTR7

(1:50, Hycult Biotech), rat anti-Crry (1:100, BD Pharmingen), rat anti-PNAd (1:100, Biolegend),

hamster anti-CD3ε (1:100 BD Pharmingen), rat anti-CD45r (1:100, BD Pharmingen), Rabbit

anti-iNOS (1:100, Calbiochem), rat anti-IDO (1:75, BD Biosciences) PE conjugated Gr1 (1:100,

Invitrogen) and Alexa 647 conjugated CD11c (1:40, Ebioscience); fluorescently conjugated

secondary antibodies were from Invitrogen, and samples were counterstained with DAPI (Vector

Laboratories).

16

In vitro characterization

Proliferation and spheroid-forming potential of tumor cell sub-lines were assessed in vitro. Cells

were seeded within 3D Matrigel matrices (BD Biosciences) in DMEM with 10% FBS, and

proliferation was measured by digesting the gel using BD Cell Recovery Solution (BD

Biosciences) at various timepoints and counting the cells. For spheroid formation studies,

cultures were maintained in 3D matrices for 6 days. In some experiments, CCL21low cells were

supplemented with 500 ng/ml rmCCL21 and control cells were treated with neutralizing

antibodies against CCR7 (20 μg/ml, Ebioscience clone 4B12). Gels were imaged and average

spheroid volume was calculated. CCR7 expression was confirmed by FACS analysis. Briefly,

murine bone marrow-derived dendritic cells (positive control) were harvested and tumor cells

were detached with Accutase then labeled with fixable live/dead violet (Molecular Probes) prior

to fixation and permeabilization (BD Cytofix Cytoperm solution, BD Biosciences). CCR7 was

then detected with anti mouse CCR7-PECy7 (Ebioscience) following the staining protocol

described earlier.

In vitro migration

A modified Boyden chamber assay was used to assess functionality of CCR7 and responsiveness

to CCL21 using 10 mm diameter, 8 μm pore transwell inserts (Millipore). 100 μl 1.8 mg/ml

collagen (BD Biosciences) containing 106 tumor cells/ml were seeded, and after polymerization,

500 ng/ml rmCCL21 (R&D Systems) was added to the lower chamber. In some chambers,

neutralizing antibodies against CCR7 (clone 4B12, R&D Systems) were added at 10 μg/ml to

both the medium chambers as well as in the gel compartment. Chambers were incubated for 24

17

h, after which gels containing non-migrated cells were removed; chambers were fixed and

membranes stained with DAPI. Migration was determined by counting 6 random fields of view.

To determine the responsiveness of immune cells to the different tumor cell sublines, tumor cells

were seeded in 24-well plates and allowed to adhere overnight, after which the medium was

switched to basal medium. Peritoneal macrophages or splenocytes were seeded into 24-well

transwell inserts, which were then incubated in the tumor cell-containing wells for 24 h.

Transmigrated cells were stained for appropriate markers and evaluated by flow cytometry.

Statistics

Unless otherwise noted, data in bar graph form are presented as mean ± SE while data shown as

individual points include the median ± S.E. Statistical significance was defined as p < 0.05

following one-way ANOVA and post-hoc analysis. When normality tests failed, Kruskal Wallis

tests were performed. Box plots are presented as Tukey box plots.

18

Supplementary Figure Legends

Figure S1: Engineered tumor cells display similar growth and CCR7 responsiveness in

vitro. (A) Determination of CCL21 secretion by ELISA of normal and tumor human cell lines

and B16-F10 melanomas in vitro. (B-E) In vitro chemotaxis of (B) CD45+ leukocytes, (C)

CD3ε+ T cells, (D) CD11c+ dendritic cells, and (E) peritoneal macrophages towards rmCCL21,

CCL21low, control, and CCL21high tumor cells. (F) CCR7 expression on CCL21low, control,

CCL21high tumor cells and murine dendritic cells by flow cytometry. (G) Tumor cell

proliferation after indicated time in 3D culture. (H and I) Representative images and

quantification of spheroid formation of tumor cells after 6 days of culture in 3D Matrigel; bar,

100 μm. (J) In vitro migration of CCL21low, control, and CCL21high tumor cells towards an

exogenous CCL21 source (500 ng/ml). No significant differences were found in CCL21-

mediated chemotaxis between tumor cell lines (P = 0.77). *P<0.05, **P < 0.01 compared with

control tumor cells.

Figure S2: Dendritic cells traffic similarly from all tumors to draining lymph nodes.

Quantification of FITC+ immune cells within draining and non tumor-draining lymph nodes

following intratumoral injection of 0.5 μm diameter FITC-labeled microspheres. (A) CD45+

leukocytes, (B) CD11c+ dendritic cells, (C) CD11c+CD86+ mature dendritic cells, and (D)

CD11c- cells isolated from lymph nodes and identified by flow cytometry (n = 3).

Figure S3: T cell subsets within B16-F10 melanomas. (A) CD3ε+ T cells expressed as a

fraction of CD45 leukocytes within CCL21low, control, and CCL21high (n ≥ 9). (B) Representative

FACS plot of intratumoral CD4+FoxP3+ TReg cell populations. (C) CD4+FoxP3+ TReg cell

populations within CCL21low, control, and CCL21high tumors expressed as % CD45+ leukocytes

(n ≥ 9).

19

Figure S4: CCL21 drives modulation of the tumor cytokine milieu. Quantification of

cytokine expression in the tumor microenvironment by ELISA (n = 7). (A) IL-2, (B) IL-4, (C)

IL-10, (D) C5a and (E) CCL2 expression in CCL21low, control, and CCL21high tumor lysates. (F)

Localization of T cells (CD3ε), B cells (CD45R/B220) and dendritic (CD11c) in CCL21low,

control and CCL21high tumors. Scale bar, 50 μm. (G) Evaluation of iNOS expression by Gr1+

MDSC cells in CCL21low, control and CCL21high tumors. Scale bar, 50 μm. Nuclei

counterstained with DAPI. Dotted line denotes tumor – dermis border. LN: lymph node, D:

dermis; T: tumor.

Fig. S5: Lymph node-like stromal networks surround CCL21-secreting tumors. (A and B)

Images and quantification of gp38+ stromal cells (green) surrounding tumors and lymph nodes.

(C) Illustration of PNAd+ CD31+ blood vessels within CCL21low, control and CCL21high tumors.

Scale bar, 50 μm. **P < 0.01 compared with control tumors.

Fig. S6: CCL21-secreting tumors attract lymphoid tissue inducer cells. Identification of

CD45+CD3-CD4+RORγt+ lymphoid tissue inducer cells (A) found within lymph nodes and

CCL21high tumors in C57BL/6 mice (n ≥ 4). Comparison of CD45+CD3-CD4+RORγt-GFP+ (B)

within lymph nodes, CCL21low tumors and control tumors in Rorc(γt)+/GFP reporter mice (n ≥ 2).

Fig. S7: Hypothesized mechanism of how tumor CCL21 promotes immune regulation. In

the early phase of tumor development, CCL21-secreting tumors attract more CCR7+ cells (naïve

T cells, iTReg cells and antigen-presenting cells) and recruit LTi cells that invoke the formation of

lymph node-like structures such as high endothelial cell-like PNAd+ vessels and gp38+

fibroblastic reticular cells that form a stromal network. This network in the tumor periphery

supports its development in several ways. For example, stromal Crry helps modulate and protect

20

21

the tumor from complement-mediated attack through inactivation of C3. Furthermore, gp38+

reticular stromal cells also secrete CCL21, which together with the structural architecture and

cytokine milieu encourages naïve T cells and DCs to co-localize, promoting regulatory

phenotypes (inset). Increased recruitment of MDSCs, likely through C5a, may inhibit T cell

effector function, while higher TGF-β1 can interfere with DC and T cell interactions and

stimulate even more TReg cells. Without this cascade of events (stroma formation, complement

regulation and TReg cell induction within a pro-tumor milieu), CCL21low tumors evoke an anti-

tumor immune response, where antigen-presenting cells, in combination with IFN-γ and IL-2

(which is not attenuated in the absence of CCR7 ligands), promote effector T cell proliferation.

MDSCs: myeloid-derived suppressor cells; iDC: immature dendritic cell; MΦ: macrophage;

TGF-β1: Transforming growth factor beta; IDO: indoleamine 2,3- dioxygenase; IFN-γ:

interferon gamma; IL-2: Interleukin-2; IL-4: Interleukin-4; Crry: Complement receptor 1–related

gene y: Rodent membrane-bound inhibitor of complement activation; CR3/4: complement

receptor 3 and 4; TCR: T cell receptor; MHC/I: major histocompatibility complex molecule

Class I; TAA: tumor associated antigen; C3: complement component 3; C3b: fragment of C3;

C5: complement component 5; LTi: lymphoid tissue inducer cell; HEV: high endothelial venule;

FRC: fibroblastic reticular cell; TReg: regulatory T cell; iTReg: inducible TReg; NK: natural killer

cell.

A

Figure S1

24 48 720

5

10

15

20 CCL21low

Control

CCL21high

Time (hours)

Cel

ls (x

104 )

G

J

MCF10a

ME275 E

PTD47

MDAMCF7

DU145ZR75

U2OS

CCL21low

contr

ol

CCL21high

0.1

0.2

0.3

0.0

CC

L21

secr

etio

n(p

g/10

00 ce

lls)

Human cell lines B16-F10

NT

rmCCL2

1

CCL21low

Contro

l

CCL21hig

h0.0

0.5

1.0

1.5

2.0

2.5*

*

**

** **

**

% m

igra

ted

cells

NT

CCL21low

Contro

l

CCL21high

2.0

3.0

0.0

1.0

Mig

ratin

g C

D3ε

+

***

NT

CCL21low

Contro

l

CCL21high

5.0

2.5

0.0

Mig

ratin

g C

D45

+ (x

104

cells

)

***

**

NT

CCL21low

Contro

l

CCL21high

1.5

1.0

0.5

0.0

Mig

ratin

g C

D11

c+ CD

11b+

*

***

B C

D

100101

102103

104CCR7

CCL21low

ControlCCL21high

BMDCs CD11c+

immature CD11c+

E F

Control CCL21high

Control+ α-CCR7

CCL21low

+rmCCL21CCL21low

Day

0D

ay 6

H

I

(x10

4 ce

lls)

(x10

3 ce

lls)

. CCL21low

0 2 4 6

0 3

0.2

0.0

0.1

Control

CCL21high

CCL21low + rmCCL21Control + α-CCR7

Days

Volu

me

(mm

3 )

B16-F

10

CCL21low

Contro

l

CCL21hig

h0.0

0.5

1.0

1.5

2.0 Control rmCCL21

**** **

**

% m

igra

ted

cells

CCR7α-n.s.

non-draining LN draining LN0.0

0.5

1.0

1.5

2.0

2.5 CCL21low

Control

CCL21high

Fluo

roB

ead+ c

ells

(x 1

03 )

non-draining LN draining LN0.0

2.0

4.0 CCL21low

Control

CCL21high

Fluo

roB

ead+ C

D86

+ ce

lls (x

102

)

non-draining LN draining LN0.0

2.5

CCL21low

Control

CCL21high

Fluo

roB

ead+ C

D11

c+

cells

(x 1

02)

non-draining LN draining LN0.0

2.5

5.0 CCL21low

Control

CCL21high

Fluo

roB

ead+ C

D11

c- cel

ls (x

102

)

A B

C D

Figure S2

5.0

CD

4

CD8α

CD

3ε

B

A

C

BA

B

0

20

40

60

80

***

CD

3ε

cells

+

(%

of C

D45

+ )

CCL21low

Contro

l

CCL21h

igh

A

Figure S3

FoxP

3

CD25

F

T TT CD

3ε C

D11

c C

D45

R

G

iNO

S G

r1 E

R-T

R7

T TT

0

1

2

3

IL-2

(pg

mg-1

)

CCL21low

Contro

l

CCL21hig

h0.0

0.5

1.0

1.5 **

IL-4

(pg

mg-1

)

CCL21low

Contro

l

CCL21hig

h0

20

40

60

80

IL-1

0 (p

g m

g-1)

CCL21low

Contro

l

CCL21hig

h

0

10

20

30

40*

CC

L2 (p

g m

l-1)

CCL21low

Contro

l

CCL21hig

h0

20

40

60 p = 0.0630

C5a

(pg

ml-1

)

CCL21low

Contro

l

CCL21hig

h

A B C

D E

Figure S4

LN CCL21low Control CCL21high

CC

L21lo

wC

CL2

1high

Con

trol

T

D

T

D

T

D

gp38 DAPI

10

20

30

40

50

0

**

**

% o

f gp3

8+ pixe

ls

B

CCL21low

Contro

l

CCL21hig

h

C

ER

-TR

7 P

NA

d

A

Figure S5

LN CCL21low Control CCL21high

Figure S6

0 10K 20K 30K

FS

0

10 0

10 1

10 2

10 3

CD

45

11.4

0 101 102 103 104

CD4

0

101

102

103

104

RORγ

t-GFP

6.17

0 101 102 103 104

CD4

010 1

10 2

10 3

10 4

CD3ε

2.26

0 10K 20K 30K

0

10 0

10 1

10 2

10 394.8

0 101 102 103 104

CD4

0

101

102

103

104

RORγ

t-GFP

C:15.4%

0 101 102 103 104

010 1

10 2

10 3

10 4

1.54

FS

CD

45

CD4

CD3ε

0 10K 20K 30K

0

10 0

10 1

10 2

10 337.6

0 101 102 103 104

CD4

0

101

102

103

104

RORγ

t-GFP

2.56

0 101 102 103 104

010 1

10 2

10 3

10 4

4.41

FS

CD

45

CD4

CD3ε

LN CCL21low ControlA

B

A

B

B

0 101 102 103

0

101

102

103

98.2

0 10K 20K 30KFS

101

102

1030 101 102 103

0

101

102

103

28.7

0.9

CD45

gp38

RO

Rγt

CD4C

D3ε

0 101 102 103

CD45

0

101

102

103

gp38

8.75

0 10K 20K 30KFS

101

102

103

RORγ

t

0 101 102 103

CD4

0

101

102

103

CD3ε

2

1.93A

A

B

B

LN CCL21highA

TGF-β

IL-4 IL-2

CCL21 High CCL21 Low

INF-γ

C3 / C5M2

Immune cell infiltration

Crry

Equilibrium

Evasion and escape

TGF-β

LTi

FRCs CrryCCL21

Figure S7

TGF-β

IDO

In situ T cell education

iNOS

iDC

LTi

DCMDSC

CCR7 Crry CR3/4

TCR MHC/I TAA

C3

C5

C3b

HEV FRC

naive T cell

iTreg (CD4+)

iTreg (CD8+)CD8+

CD4+naturalTreg

NK

CCL21 gradient

effectorCD8+

migration

MΦ