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: [email protected]
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