supplemental table 1. primer se quences for transcript ... · fsc-a ly6c gr-1 ly6c ccr2 ccr2 ly6c...

Supplemental Table 1. Primer sequences for transcript analysis

Abbreviations: Mmp, matrix metalloproteinase; Timp, tissue inhibitor of metalloproteinase; Col, collagen; Fn, fibronectin; Ccl, chemokine (C-C motif) ligand

Primer Sequence (5’ – 3’) Primer Sequence (5’ – 3’)

Mmp2 Forward ACCCAGATGTGGCCAACTAC Mmp15 Forward CAGGAAAGGCATGGAACAATReverse AAAGCATCATCCACGGTTTC Reverse ACCAATGGTGTGACCTGCTC

Mmp3 Forward TGGAGATGCTCACTTTGACG β-Actin Forward CAGCTTCTTTGCAGCTCCTTReverse AGCCTTGGCTGAGTGGTAGA Reverse CACGATGGAGGGGAATACAG

Mmp7 Forward CGGAGATGCTCACTTTGACA Timp1 Forward GCATCTGGCATCCTCTTGTT Reverse ACCGGGAACAGAAGAGTGAC Reverse TGGGGAACCCATGAATTTAG

Mmp8 Forward CTTTCAACCAGGCCAAGGTA Timp2 Forward AAGCAGTGAGCGAGAAGGAG Reverse GAGCAGCCACGAGAAATAGG Reverse GGGGGCCGTGTAGATAAACT

Mmp9 Forward AGACGACATAGACGGCATCC Timp3 Forward GTGGGAAAGAAGCTGGTGAA Reverse TGGGACACATAGTGGGAGGT Reverse AGAGGCTTCCGTGTGAATGT

Mmp10 Forward CAGTTGGAGAACACGGAGAC Col1a1 Forward TAAGGGTACCGCTGGAGAAC Reverse TGTCCATTTCTCATCATCATCG Reverse CTCCCTGAGCTCCAGCTTCT

Mmp11 Forward GACGCTGGGAGAAGACAGAC Col1a2 Forward AGAGGACCACGTGGACAAAG Reverse GTGGGGTCACTTCACTCCATA Reverse TGAGCAGCAAAGTTCCCAGT

Mmp12 Forward GCTGTCACAACAGTGGGAGA Fn Forward TTAAGCTCACATGCCAGTGCReverse ATACCAGATGGGATGCTTGG Reverse CCCACTTCTCTCCGATCTTG

Mmp13 Forward TGGACCTTCTGGTCTTCTGG Ccl2 Forward AGGTCCCTGTCATGCTTCTGReverse TGGGCAGCAACAATAAACAA Reverse ATTGGGATCATCTTGCTGGT

Mmp14 Forward CCAGTGGATGGACACAGAGAReverse AGAGGGCCGAGAGGTAGTTC

Supplemental Figure 1

InflammatoryMonocytes

ResidentMonocytes Granulocytes

SSC

-A

CD45

SSC

-A

CD19 F4/80

FSC

-A

Ly6C

Ly6G

Ly6C

CCR2CCR2

Ly6C

Ly6G

Ly6C

A


InflammatoryMonocytes

ResidentMonocytes

Granulocytes

SSC

-A

CD45

SSC

-A

CD19 F4/80

FSC

-A

Ly6C

Gr-1

Ly6C

CCR2CCR2

Ly6C

Gr-1

Ly6C

Supplemental Figure 1. Gating strategy for identification of monocyte subpopulations. Fluorescence-activated cell sorting(FACS) plots showing the gating strategy for identifying resident monocytes, inflammatory monocytes and granulocytes usingantibodies that recognize (A) Ly6G or (B) Gr-1 in combination with antibodies that recognize CD45, CD19, F4/80, Ly6C and CCR2.

CCR2

Ly6C

B


BA

72 hr

analysis 0

1

2

3

4

5

Ctrl

anti-CD40

10 g 30 g 100 g

Abs

olut

e nu

mbe

r of

F4/

80+

mac

roph

ages

(x10

6 )

*****

Supplemental Figure 2. CD40 agonist induces accumulation of resident macrophages in spleen. a, Wild-type C57BL/6 mice were administered isotype control or anti-CD40 antibody (0.1 mg) by intraperitoneal injection. b, Quantification of the absolute number of F4/80+ macrophages within spleens of mice after treatment with control or escalating doses of anti-CD40. n = 3 mice per group. **P< 0.01, ***P <0.001, unpaired t-test.

Ly6GDAPI

Ctrl FGK45


Supplemental Figure 3. Impact of anti-CD40 treatment on Ly6G+ cells within PDAC tumors of KPC mice. Tumor-bearing KPC mice were treated with isotype control (Ctrl) or anti-CD40 antibodies. One day later, tumors were analyzed by immunofluorescencemicroscopy for the presence of Ly6G+ cells (green). Nuclei are stained with DAPI (blue). Displayed are representative images with quantification shown in Figure 1H.


B

D E

ALy

6C

FSC

PreDay +1

post CEL

IMRM

Supplemental Figure 4. Strategy for depletion of resident and inflammatory monocyte populations in vivo. FACS plots gated on CD45+ CD19neg F4/80+ cells showing resident (RM) and inflammatory (IM) monocytes pre and one day post treatment with a,clodronate encapsulated liposomes (CEL), b, anti-Gr-1 depleting antibody, RB6-8C5, and c, anti-Ly6C depleting antibody, Monts-1. d, Quantification of absolute number of resident monocytes in peripheral blood pre and post treatment with clodronate encapsulated liposomes. n=5 mice, P = 0.002, paired t-test. e, Quantification of absolute number of inflammatory monocytes in peripheral blood pre and post treatment with RB6-8C5 (anti-Gr-1). n=7 mice, P=0.002, paired t-test. f, Quantification of inflammatory monocytes in peripheral blood pre and post treatment with Monts-1 (anti-Ly6C). n=5 mice, P=0.009, paired t-test.

PreDay +1

post anti-Gr-1

Ly6C

FSCIMRM

C

F

PreDay +1

post anti-Ly6C

Ly6C

FSCIMRM

Day of CEL treatmentPre Day 1

# of

cel

ls/1

0µl o

f blo

od

4000

300020001000

5000

0

Day of anti-Ly6C treatmentPre Day 1

# of

cel

ls/1

0µl o

f blo

od

4000

300020001000

5000

0

Day of anti-Gr-1 treatmentPre Day 1

# of

cel

ls/1

0µl o

f blo

od4000

300020001000

5000

0

RM IM IM

0

20000

40000

60000

Day of CEL treatment

Pre Day 1

# of

cel

ls/1

0µl o

f blo

od

0

2000

4000

6000

8000

10000

Pre Day 1


# of

cel

ls/1

0µl o

f blo

od

F4/80+Ly6Clo

RMF4/80+Ly6Chi

IMF4/80neg

Ly6C+ Ly6G+

0

1000

2000

3000

4000

Pre Day 1


# of

cel

ls/1

0µl o

f blo

od


A B C

Supplemental Figure 5. Effect of clodronate encapsulated liposome (CEL) treatment on peripheral blood myeloid cells. Shown are peripheral blood counts for (a) F4/80+ Ly6Clo resident monocytes (RM) , (b) F4/80+Ly6Chi inflammatory monocytes (IM) and (c) F4/80neg

Ly6C+ Ly6G+ cells prior to (Pre) and one day (Day 1) post treatment with CEL. The impact of CEL on F4/80+Ly6Clo resident monocytes(RM) displayed in (a) is also shown in Supplemental Figure 4D and depicted here for comparison purposes. Significance testing was performed using a paired t-test. n=5; F4/80+ Ly6Clo RM, P= 0.002; F4/80+ Ly6Chi IM, P= 0.871; F4/80neg Ly6C+ Ly6G+, P =0.113.

0

10

20

30

40

0

50

100

0.00

0.10

0.20

0.30

0.0

0.5

1.0

1.5

0

5

10

15


A

B

Controlanti-Ly6C

anti-CD40anti-CD40 + anti-Ly6C

Tota

l cel

l num

ber

(x10

6 )

Tota

l cel

l num

ber

(x10

6 )

Tota

l cel

l num

ber

(x10

6 )

Tota

l cel

l num

ber

(x10

6 )

Tota

l cel

l num

ber

(x10

6 )

CD3+ T cells CD19+ T cellsF4/80+ Ly6Chi

IMF4/80+ Ly6Clo

RMF4/80neg

Ly6C+ Ly6G+

* * *

*

**

050

100150200

Cel

ls/

Lbl

ood

0100200300400

Cel

ls/

Lbl

ood

0500

100015002000

Cel

ls/

Lbl

ood***

Supplemental Figure 6. Specificity of Ly6C depleting antibody, Monts-1. a, Peripheral blood counts for F4/80+ Ly6Chi

inflammatory monocytes (IM) , F4/80+ Ly6Clo resident monocytes (RM) , and F4/80neg Ly6C+ Ly6G+ cells prior to (Pre) and one day after (Day +1) treatment with anti-Ly6C depleting antibody, Monts-1. n=8 mice, paired t-test. b, Mice were treated with or without anti-Ly6C on days -1 and 0 with anti-CD40 administered on day 0. Splenocytes were harvested on day +1 and absolute number of CD3+ T cells, CD19+ B cells, F4/80+ Ly6Chi IMs, F4/80+ Ly6Clo RMs, and F4/80neg Ly6C+ Ly6G+ cells were quantified by flow cytometry. n = 4 mice/group, 2-tailed unpaired t-test. *P<0.05, **P<0.01, ***P<0.001.

F4/80+ Ly6Chi

IMF4/80+ Ly6Clo

RMF4/80neg

Ly6C+ Ly6G+

A B


Control anti-CD40

anti-CD40 + CEL Anti-CD40 + anti-CCL2

Supplemental Figure 7. CCL2 regulates CD40-dependent Ly6C+ myeloid cell recruitment to PDAC tumors in KPC mice.a, Representative images of immunohistochemistry to detect Ly6C+ cells in PDAC tumors from KPC mice one day aftertreatment with isotype control or anti-CD40 antibodies with or without clodronate encapsulated liposomes (CEL) or anti-CCL2neutralizing antibodies. Quantification is shown in Figure 2C. b, CCL2 protein levels detected by cytometric bead array inpancreas of normal littermate (n=2), subcutaneously (SubQ) implanted PDAC tumors (n=3), and primary PDAC tumors isolatedfrom KPC mice (n=4).

0

1

2

3

ng C

CL2

/gra

m ti

ssue

Normalpancreas

SubQtumor

KPCtumor


Supplemental Figure 8. Anti-CD40 treatment induces systemic cytokine release in mice and humans. a, Wild-type non-tumorbearing mice and KPC tumor-bearing mice were treated with isotype control (Ctrl) or anti-CD40 antibody. Serum was collected 24 hoursafter treatment for analysis. Shown are serum levels (pg/mL) of IL-12. n=17-20 mice per group for wild-type and n=10 mice per groupfor KPC. b, KPC tumor-bearing mice were treated with Ctrl, anti-CD40 or anti-CD40 in combination with anti-IFN- neutralizingantibody. Shown are serum levels of IL-12 and IFN- detected one day after treatment. n=5-14 mice per group. For a and b, significancetesting was performed using Student’s 2-tailed unpaired t-test. c, Shown are plasma levels for IL-12, IP-10, and MIG in PDAC patientsPre and one day post treatment with CP-870,893. For detection of plasma cytokine levels, n=13 patients at baseline; n=5 patients afterCP-870,893. Significance determined using Mann-Whitney test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

A B

****

****

pg/m

L

1500

1000

500

0

pg/m

LIL-12 IFN-

Ctrlanti-CD40anti-CD40 + anti-IFN-

***

******

C

2000

1500

1000

0

500

Ctrl anti-CD40

Ctrl anti-CD40

Wild-type KPC

IL-12

2000

4000

6000

pg/m

L

0

Day +1

Pre

****

*

Day 0

Day 1

Day 2

Day 5

pSTAT1


MFI: 265

MFI: 478

MFI: 265

MFI: 254

Supplemental Figure 9. STAT1 signaling in human monocytes induced by plasma from PDAC patient treated with CP-870,893. Kinetics of (a) IL-12 and (b) IFN-detected in plasma of PDAC patients prior to and after treatment with the fully human CD40 agonist, CP-870,893. n=5-13 patients per time point. b, Human monocytes were incubated with plasma collected from a patient at day 0 (prior to treatment) and on days 1, 2, and 5 post treatment with CP-870,893. Monocytes were evaluated by flow cytometry for pSTAT1 expression. Shown are representative histograms of monocytes obtained from one of three healthy volunteers. Quantification of results is shown in Figure 3D. MFI, mean fluorescence intensity. For panels a and b, significance testing was performed using one-way ANOVA with Bonferroni correction. **, P<0.01; ****, P<0.0001.

pg/m

Lpg

/mL

IL-12

IFN-

Day

Day

A

B

C

4000

3000

2000

1000

00 1 2 5 12 25

150

100

50

00 1 2 5 12 25

****

**

Supplemental Figure 10. Anti-fibrotic activity induced with a CD40 agonist is dependent on IFN-. Representative images of tumorsections analyzed by H&E and by Masson's trichrome stain to reveal extracellular matrix (blue) one day after treatment. n=5-6 mice pergroup. Scale bar, 50 µm.

Ctrl

anti-CD40

anti-CD40 + anti-IFN-

H&EMasson’sTrichrome


Supplemental Figure 11. CD40 activation does not alter extracellular matrix production or FAP+ fibroblast proliferation.Syngeneic mice bearing established PDAC tumors implanted subcutaneously were treated with isotype control or anti-CD40 antibodies.Tissues were harvested and analyzed one day later. a, Shown is relative transcript expression with comparison to -actin for Col1a1,Col1a2, and Fn observed within tumor homogenates. Data are shown as means + s.d. from n=5 mice for each transcript. b,Quantification of tumor sections for Ki67+ FAP+ cells by immunofluorescence microscopy. c, Representative immunofluorescenceimages of Ki67 (green) and FAP (red) staining in tumor tissues. Nuclei were stained with DAPI (blue). Scale bar, 100 m.


Col1a1 Col1a2 Fn

150

100

50

0

Rel

ativ

e tr

ansc

ript

expr

essi

on

Ctrlanti-CD40

A B

Ki6

7+FA

P+ce

lls p

er h

pf

0

0.5

1.0

1.5

2.0

CKi67 FAP DAPI

Ctrl

anti-CD40


Supplemental Figure 12. Anti-CD40 treatment selectively depletes extracellular matrix proteins within the tumormicroenvironment. a, Representative images of H&E and immunofluorescence for type I collagen (TIC) and fibronectin (FN)deposition (red) among EpCAM+ tumor cells (green) one day after treatment with isotype control (Ctrl) or anti-CD40 antibodies. b,Representative images of immunohistochemistry for hyaluronan (HA) one day after treatment with isotype control (Ctrl) or anti-CD40antibodies.

Ctrl

anti-CD40

TICEpCAM

FNEpCAMH&E

HA

Ctrl

anti-CD40

A B

Supplemental Figure 13. Implantable tumor model of PDAC that reproduces tumor microenvironment of KPC model ofspontaneous PDAC. Representative images of H&E (A, D), Masson’s trichrome staining (B, E) and immunohistochemistry forF4/80+ macrophages (C, F) are shown for a spontaneously arising PDAC tumor in the KPC model (A-C) and a cell line derivedfrom this tumor that was implanted subcutaneously into C57BL/6 syngeneic mice (D-F).


D E F

A B C

A B

C D50

40

30

20

10

0Ctrl anti-CD40

Rel

ativ

e %

Are

a

Type I Collagen

*80

60

40

20

0

# of

IM c

ells

per

hpf

Ctrl anti-CD40

*

2 weeks

analysisC57BL/6

24 hrs

anti-CD40Ctrl

IM

Type I Collagen


Supplemental Figure 14. Development of an implantable model of PDAC for evaluating monocyte-dependentdegradation of cancer fibrosis. a, Schematic showing treatment of C57BL/6 mice implanted subcutaneously with a syngeneicPDAC cell line derived from KPC mice. At two weeks after tumor implantation, mice were treated with isotype control or anti-CD40. The impact of treatment on the tumor microenvironment was examined 24 hours later. b, Tumors were analyzed forLy6C+ inflammatory monocyte (IM) infiltration by staining for Ly6C and for changes in extracellular matrix deposition bystaining for type I collagen. One representative image out of 4 is shown per group. Scale bar, 50 m. c, Quantification of tumor-infiltrating Ly6C+ inflammatory monocytes by immunohistochemistry. *, P < 0.05; unpaired 2-tailed t-test determined from 5-6images per mouse. d, Quantification of type I collagen content. *, P < 0.05; unpaired 2-tailed t-test determined from 4 imagesper mouse, n=4-5 mice per group.

Ant

i-CD

40 in

duce

d ex

pres

sion

leve

ls

rela

tive

to C

ontr

ol (

C

T)A

nti-C

D40

indu

ced

expr

essi

on le

vels

re

lativ

e to

Con

trol

(

CT)

ECM ProteinsCollagensAdhesion MoleculesAdamts

Integrins Laminins MMPs and TIMPs Other Proteins

Supplemental Figure 15. Gene expression of extracellular matrix and adhesion molecules within the tumor microenvironment inresponse to anti-CD40 treatment. Fold change in gene expression within tumor homogenates one day after treatment with anti-CD40antibodies compared to isotype control.


A B

Rel

ativ

e tr

ansc

ript

expr

essi

on

0

0.05

0.10

0.25

0.15

0.20

Rel

ativ

e tr

ansc

ript

expr

essi

on0

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40

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60

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Rel

ativ

e tr

ansc

ript

expr

essi

on

0

2

4

10

6

8

Ctrl Mo Mo+ IFN-

Ctrl Mo Mo+ IFN-

Ctrl Mo Mo+ IFN-

*

C


Supplemental Figure 16. IFN- induces Mmp13 expression in myeloid cells. Murine bone marrow derived myeloid cellswere stimulated in vitro with or without IFN- and then plated on a matrix of type I collagen containing human PDAC cells.Murine-specific Mmp gene expression was detected 24 hours after addition of unstimulated myeloid cells (Mo), IFN-stimulated myeloid cells (Mo + IFN-), and no addition of myeloid cells (Ctrl). Shown is the relative transcript expression for(a) Mmp10, (b) Mmp12, and (c) Mmp13. Data are shown as means + s.d., from 3 independent experiments. *, P<0.05.

Mmp10 Mmp12 Mmp13


Supplemental Figure 17. RNA in situ hybridization (ISH) for MMP13 in PDAC tumors. Representative images of RNAISH for Mmp13 in spontaneously arising PDAC tumors in KPC mice one day after treatment with isotype control or anti-CD40antibody. n=3 mice per group, 5 images per mouse.

Control Anti-CD40


A

Supplemental Figure 18. Detection of MMP activity in cellular subsets within the tumor microenvironment. Treatmentschema for detection of MMP activity. a, C57BL/6 mice were injected subcutaneously with PDAC tumor cells and 14 days latermice were administered MMPsense or PBS at three hours prior to treatment with Ctrl or anti-CD40. At 24 hours later, tumorswere evaluated by flow cytometry for presence of active MMPs. b, Flow cytometric analysis comparing cellular subsets withintumor tissue for the presence of MMP activity. n = 4-5 mice per group.

analysis

24 hours

MMPsenseor PBS

B

Total CD45+ CD45neg0

10203040

% c

ells

with

ac

tive

MM

Ps

F4/80+Ly6Chi Ly6G+0

1020304050 Ctrl

anti-CD40

A B C


Supplemental Figure 19. Protein expression of MMP13 and MMP14 within the tumor microenvironment ofspontaneously arising pancreatic tumors in the KPC model. Representative immunofluorescence images with arrowsindicating a, MMP13 (green) co-localization with F4/80+ (red) myeloid cells (scale bar, 10 m) and b, MMP13 (green) co-localization with FAP+ (red) fibroblasts (scale bar, 20 m) c, Expression of MMP14 (green) by F4/80+ (red) myeloid cells withinstroma but not in tumor cells (T) of KPC mice one day after treatment with isotype control (Ctrl) or anti-CD40 antibody.

MMP14F4/80

Ctr

lan

ti-C

D40

MMP13F4/80

MMP13FAP

TT

TT

T


Supplemental Figure 20. Gene expression of Mmp2 and Mmp14 within the tumor microenvironment of pancreatictumors. Shown is the relative transcript expression with comparison to -actin for Mmp2 and Mmp14 observed within tumorhomogenates one day after treatment (Ctrl versus anti-CD40) of syngeneic mice bearing established PDAC tumors implantedsubcutaneously. Data are shown as means + s.d., from n = 9-10 mice for each transcript.

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

Ctrl Anti-CD40

Rel

ativ

e ex

pres

sion

Mmp2Mmp14

FN EpCAM DAPI


Supplemental Figure 21. Anti-fibrotic activity of a CD40 agonist is dependent on MMPs. a, Schematic showing treatment of miceimplanted subcutaneously with a syngeneic PDAC cell line derived from KPC mice. At two weeks after tumor implantation, mice weretreated with a broad spectrum MMP inhibitor (iMMP, Actinonin) or PBS followed 30 minutes later by isotype control or anti-CD40antibodies. b, Representative immunofluorescence images show fibronectin (FN, red) deposition among EpCAM+ tumor cells (green) oneday after treatment. Nuclei are stained with DAPI (blue). One representative image is shown out of five images per tumor section, pergroup. Scale bar, 100 m.

2 weeks

analysisC57BL/6

A B

30 min 24 hrs

Masson’sTrichrome

Ctrl anti-CD40anti-CD40 +iMMP13 #1

anti-CD40 +iMMP13 #2

Anti-CD40 +iMMP


Supplemental Figure 22. Impact of MMP13 specific inhibitors on CD40 agonist induced collagen degradation. Mice with animplanted PDAC tumor were treated with isotype control or anti-CD40 antibodies with or without selective inhibitors of MMP13(iMMP13 #1, WAY-170523; iMMP13 #2, 544678-85-5) or a broad spectrum MMP inhibitor (iMMP, Actinonin). Representative imagesshow collagen deposition (blue) by Masson’s Trichrome staining one day after treatment. One representative image is shown out of fiveimages per tumor section, n=4-8 tumors per group.


Con

trol

Day

1D

ay 2

Day

4D

ay 7

A B

0

0.2

0.4

0.6

0.8

1

1.2

Ctrl 1 2 4 7

Day post FGK45

Rat

io E

B le

akag

e(tu

mor

:kid

ney)

*

C

4x

20x

Ctrl FGK45 (d+2)

Supplemental Figure 23. Kinetics of anti-CD40 activity on tumor microenvironment. Mice with implanted tumors were analyzed atdefined time points after treatment with isotype control or anti-CD40 antibodies. a, Gross images of bisected tumors analyzed at indicatedtime points after anti-CD40 treatment with comparison to isotype control. b, Shown is the ratio of Evans Blue (EB) leakage into tumortissue normalized to kidney. Evans blue was administered 20 minutes prior to necropsy at defined time points after treatment with anti-CD40 or isotype control antibody. c, Representative H&E images (4x and 20x) showing hemorrhagic necrosis in tumors at day 2 afteranti-CD40 treatment compared to isotype control. n=7-8 tumors per treatment group, *, P<0.05.


A B

Day post anti-CD400 1 2 4 7

0

5

10

15

20

CD

31+

vess

els

per h

pf

Ctrl

anti-CD40(d+2)

CD31

Supplemental Figure 24. Impact of anti-CD40 on CD31+ blood vessel density and vascular patency. Mice with implanted tumorswere analyzed on multiple days after treatment with isotype control or anti-CD40 antibodies. a, Shown is the density of CD31+ vesselsper hpf. n=7-8 tumors per group, 5 images per tumor. b, Representative images showing collapsed CD31+ blood vessels within tumors.


Ctrl

anti-CD40 Gem (d+2)

Gem

anti-CD40 Gem (d+5)analysis

C57BL/624 hrs2 or 5 days2 weeks

Supplemental Figure 25. Anti-CD40 treatment improves gemcitabine efficacy in vivo. a, Schematic showing treatment of miceimplanted subcutaneously with a syngeneic PDAC cell line derived from KPC mice. At two weeks after tumor implantation, mice weretreated with anti-CD40 antibodies or control (Ctrl) followed by gemcitabine administered two (d+2) or five days (d+5) later. One day posttreatment with gemcitabine, tumors were analyzed for the number of Ki67+ cells per hpf. b, Representative images showing detection ofKi67+ cells within tumors from indicated treatment groups. Quantification is shown in Figure 6D.

A B


Gem anti-CD40 Gem (d+2)

A

Ki67

B

**

0

20

40

60

80

Ki6

7+ce

lls p

er h

pf

Gem anti-CD40 Gem (d+2)

Supplemental Figure 26. Anti-CD40 treatment improves gemcitabine efficacy in KPC tumor-bearing mice. KPC mice were treatedwith anti-CD40 antibodies or control followed two days later by gemcitabine. One day post treatment with gemcitabine, tumors wereanalyzed for the number of Ki67+ cells per hpf. a, Representative images showing detection of Ki67+ cells within tumors from indicatedtreatment groups. b, Quantification of Ki67+ cells per hpf. n=3-4 mice per group, 5 images per mouse. **, P<0.01, student’s ttest.


0

1000

2000

-50

-100% C

hang

e in

tum

or v

olum

e

***

**

Supplemental Figure 27. Impact of MMPs and Ly6C+ cells on anti-tumor activity induced by gemcitabine administered two daysafter an agonist CD40 antibody. Mice implanted subcutaneously with a syngeneic PDAC cell line derived from KPC mice were treatedwith anti-CD40 or control with or without inhibition of MMPs (iMMP, Actinonin) or depletion of Ly6C+ cells. Two days after anti-CD40treatment, mice received gemcitabine. Shown is the relative change in tumor volume from time of gemcitabine treatment to 6 days later.n=6-13 mice per group. *, P<0.05, **, P<0.01, Student’s t test.

Day post Gem treatment

0 1 2 3 4 5 6

Perc

ent c

hang

e in

bod

y w

eigh

t

15

10

0

-10

-15

-5

5


****

Supplemental Figure 28. Treatment tolerability is dependent on timing of gemcitabine administration after anti-CD40 treatment.Non-tumor-bearing mice were treated with anti-CD40 or control antibodies followed by gemcitabine administered two (day +2) or five days (day +5) later. Shown is the percent change in body weight over time. n=4-8 mice per group. Significance testing was performed using one-way Anova with Bonferroni correction for multiple comparisons testing. Error bars represent standard error of the mean, ** P<0.01.

Gem

anti-CD40 Gem (day +5)anti-CD40 Gem (day +2)

supplemental table 1. primer se quences for transcript ... · fsc-a ly6c gr-1 ly6c ccr2 ccr2 ly6c...

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