Supplemental Fig 1

Rt. Lung

Tumor Tumor

A B

Murine lung cancer model. LLC were implanted as described in Fig 1. A) Representative histology showing the distribution of tumor nodules on day 24. B) Representative H&E stained photomicrograph showing a tumor nodule (Bar = 500 μm).

Supplemental Fig 2

B

Free CpG ODN (ng)

37.5

CpG on microparticles

75 150

300

600

900

1200

1800

0 hr

3 hr

24 h

r48

hr

A

0 12 24 36 480

20

40

60

80

100

%O

DN

re

lea

se

Time (hr)

C

1 μm

Release of CpG ODN from CpG-MP. (A) Scanning electron photomicrograph showing a microparticle assembled after freeze-drying. CpG-MP were suspended in saline just before scanning. Bar = 1 μm. (B) Desorption of CpG ODN from CpG-MP was monitored by suspending particles in physiological saline (pH 7.4, 37o C). Supernatants were collected over time, loaded onto a 3% agarose gel, electrophoresed and visualized by staining with SYBR Gold. (C) The percent of ODN released was calculated by the formula: (measured ODN)/(total ODN used to formulate the CpG-MP) x100%. Results are representative of 3 independent experiments.

Supplemental Fig 3S

urv

iva

l (%

)

Day

TLR9 KO Rag1 KO

**

No Rx (WT)

CpG-MP i.t. No Rx

Day

Effect of CpG-MP on survival of LLC challenged mice. LLC were implanted and mice treated with CpG-MP as described in Fig 1.

Survival curves were generated for TLR9 KO and Rag1 KO mice and analyzed by Kaplan-Meier statistics using the log-rank test. Data from 2-3 independent experiments involving 7-10 mice/group were combined to generate each survival curve. **, p < 0.01 vs No Rx (WT) (Kaplan Meier).

Supplemental Fig 4Untreated CpG-MP treated

CD8 T cells

M2-like macrophages

Effect of CpG-MP on immune cells in LLC tumor. LLC were implanted and mice treated with CpG-MP as described in Fig 1.

Lungs were collected on day 20. Photomicrographs compare portions of tumors from untreated vs CpG-MP treated mice. Sections were stained to detect CD3+, CD8+ T cells (A,B) or F4/80+, CD206+ M2 macrophages (C,D). Bar = 50 μm.

A B

C D

Supplemental Fig 5 A

Rx on Day 18 Rx on Days 7, 14

No Rx

B#

of

Ap

op

toti

c c

ells

100 101 102 103 104

# of CTL

p < 0.01r = 0.79

102 103 104

# o

f A

po

pto

tic

ce

llsImmunosuppressive

cell #

p < 0.01r = - 0.36

Correlation between cancer cell apoptosis and tumor infiltration by CTL vs immunosuppressive cells. LLC were implanted and mice treated as described in Fig 6. Lungs were collected on day 20. The number of apoptotic (TUNEL+) tumor cells is plotted against the number of A) CD3+ CD8+ CTL and (B) immunosuppressive cells (M2 macrophages plus Tregs) per mm2 of tumor. Data were derived by analyzing serial sections from a total of 15-39 tumors in 3 mice per group (One way Anova).