Endothelin (B) Receptor Mediates the Endothelial Barrier to T-cell Homing to Tumors and Disables Immune Therapy Ronald J. Buckanovich, Andrea Facciabene, Sarah Kim, Fabian Benencia, Dimitra Sasaroli, Klara Balint, Dionysios Katsaros, Anne O’Brien-Jenkins, Phyllis A. Gimotty, and George Coukos Supplemental Figure 1

Figure S1

Endothelin-1 (ET-1) mRNA expression in ovarian cancer with or without TIL (n=16

each, mean ±SD, p=0.26).

Supplemental Figure 2

Figure S2

Effect of TIL on overall survival (A) and disease-free survival (B) in the same patients as

in Figure 2g. Black, Patients with tumors exhibiting TIL (n=32 in A, n=31 in B); Red,

Patients with tumors exhibiting no TIL (n=25 in A, n=22 in B).

Supplemental Figure 3

Figure S3

Effect of ETBR expression and TIL on overall survival (A) and disease-free survival (B)

in the same patients as in Figure 2g of manuscript. Blue, ETBR low and TIL present;

Red, ETBR low and TIL absent; Black, ETBR high and TIL absent; and Green, ETBR

high and TIL present. Three year disease-free interval (DFI) and overall survival rates are

reported in the Table underneath.

Supplemental Figure 4

Figure S4

Distinct effects of ETAR and ETBR blockade in vitro. A, ETBR antagonist BQ-788

increases ICAM-1mRNA (A) or protein expression (B), while ETAR antagonist BQ-123

does not.

Supplemental Figure 5

Figure S5

ICAM-I and ETBR mRNA expression quantified in 16 tumors with TIL and 16 tumors

without TIL (mean ±SD). ICAM-1 mRNA levels are approximately two fold higher in

tumors with TIL relative to tumors lacking TIL (p=0.06). A reciprocal relationship can be

seen between expression of ICAM-1 and ETBR.

Supplemental Figure 6

Figure S6

ETBR antagonist BQ-788 does not induce inflammation in liver or kidneys of vaccinated

mice. A. H&E of liver and kidney of mice receiving vaccine + BQ-788 peptide, vaccine,

or no treatment (CTRL). B. CD45 IHC in liver and kidney of mice receiving vaccine or

vaccine + BQ-78 peptide. Top, panels show representative results; bottom,

quantification of cell counts in ten 40X fields shows no significant difference in CD45+

cells infiltrating the kidney or the liver in mice receiving vaccine or vaccine + BQ-788

peptide.

Supplemental Figure 7

Figure S7

ETBR antagonist BQ-788 does not increase T cell homing to lung, spleen, liver or kidney

in mice receiving adoptive transfer of T cells or a therapeutic vaccine. A. Detection of

CFSE-labeled T cells transferred adoptively to mice with or without combination with

BQ-788 therapy. Histograms show relative fluorescence units counted in ten 40X fields

(mean ±SD). B, CD3 IHC reveals T cells in liver and kidney of mice receiving vaccine,

vaccine + BQ-788, or no treatment. Top, panels show representative results; bottom,

quantification of CD3+ cell counts in ten 40X fields.

Supplemental Table 1 Human Primers ETBR fwd 5’-AGAAAAGTGGCATGCAGATTG-3'

rev 5’-AGAATCCTGCTGAGGTGAAGG-3' KIAA1699 fwd 5’-ACGTGCACCACGTACAACAG-3' rev 5’-ATCCAGGATGCTCTTGTTGC-3' NCOA1 fwd 5’-GGCAGGTGGAAATACGAATG-3' rev 5’-GTGCGGGATCATTTATCTGC-3' ADRBK2 fwd 5’-AAGAAGCCTCATGCGAGTGT-3' rev 5’-CAGAAGTTTGAAAAGCATGCAG-3' CALD1 fwd 5’-CAAGTCACCTGCTCCCAAAC-3' rev 5’-TGAGCTTGGGTTCTTTCTGG-3' COMPLEMENT-3 fwd 5'-ACACTACCTCATGTGGGGTCTC-3' rev 5’-GGCATTGTTTCTGGTTCTCTT C-3' ESR1 fwd 5’-TCCTCATCCTCTCCCACATC-3' rev 5’-CCAGCAGCAGGTCATAGAGG-3' HSPCO56 fwd 5’-TGACCCTGGTAAATGTTTTGG-3' rev 5’-ATCTGTCCGAATTGCTCCAG-3' BETA-ACTIN fwd 5'-ACACAGGGGAGGTGATAGCATT-3' rev 5’-ATACATCTCAAGTTGGGGGACAA-3' ICAM-1 fwd 5’-AGCACTCAAGGGGAGGTCAC-3' rev 5’-CCTGCAGTGCCCATTATGAC-3' ICAM-2 fwd 5’-AACACTCAGCCCCGAAGATG-3' rev 5’-CCGAAGATGAAGCAGAGCAG-3' ICAM-3 fwd 5’-GGATCCCGTTCTTCGTCAAC-3' rev 5’-CGAAGACGGGGACAAAGTG-3' E-SELECTIN fwd 5’-GCCTTGAATCAGACGGAAGC-3' rev 5’-GTAGGCAAGAAGGGCCAGAG-3' JAM fwd 5’-ATAGCCGAGGCCACTTTGAC-3' rev 5’-AGGCTCACACCAGGAATGAC-3' CXCL11 fwd 5’-GCCTTGGCTGTGATATTGTGT-3' rev 5’-CTTTTACCCCAGGGCCTATG-3' LFA-1 ALPHA fwd 5’-TCATGAAGGTTGACGTGGTG-3' rev 5’-TCTCCTTCAGGTTCCGTTTG-3' LFA-1 BETA fwd 5’-GGATGGACCGCTACCTCATC-3' rev 5’-TCAGAGCCTTCCAGATGACC-3' L-SELECTIN fwd 5’-ACCCCCTCTTCATTCCAGTG-3' rev 5’-CACCAAGGGCGATTTAATATG-3' Mouse Primers IL-2 fwd 5'-TGGAGCAGCTGTTGATGGAC-3'

rev 5'- GCC TGC TTG GGC AAG TAA AA-3' IFN-gamma fwd 5’-TGG AGG AAC TGG CAA AAG GA-3’

rev 5’-TGT TGC TGA TGG CCT GAT TG-3’

SUPPLEMENTAL METHODS

Patient Tissues

Frozen stage-III epithelial ovarian cancer samples for immunohistochemistry-guided laser

capture microdissection (immuno-LCM), western blot, qRT-PCR and survival analyses

were from the University of Turin, Italy. Fresh tumor samples for cell purification and/or

molecular studies were collected at the University of Pennsylvania.

Immuno-LCM, array hybridization and analysis

We used Microcut (MMI) to isolate up to 2 x 103 immunopositive cells per specimen.

RNA was extracted using the TRIzol microprotocol and amplified twice using the Ambion

MessageAmp kit. cRNA was biotin-labeled (12-24h, ENZO RNA labeling kit); purified

using RNA cleanup (Qiagen); hybridized to U133A and U133B Affymetrix arrays; and

scanned as recommended in the Affymetrix GeneChip® protocol (www.Affymetrix.com),

as previously reported1. All chips were scanned twice using MAS 5.0 with a normalization

factor of 1.0 and target intensity of 150. Median scaling factor was 2.9 (range 1.45-10; a

higher than normal scaling factor was tolerated for some samples given the amplification

process). Arrays were reproducible with correlation r2 0.89-0.94. To identify genes with

overexpressed in endothelial cells from TIL-positive vs. TIL-negative tumors we required a

gene to be ‘present’ (determined by MAS 5.0 Suite, Affymetrix) in at least 6 of 11 TIL-

negative samples. To identify genes induced in overexpressed in TEC from TIL-positive

tumors we required genes to be present in at least 9 of 14 TIL-positive samples. All

samples were normalized with the median defined as 1.0. Analysis of expression profiles

used Genespring software (Agilent). For each gene, we characterized the fold change

defined as the ratio of the normalized mean expression of the gene in TEC from tumors

with TIL to its expression in tumors lacking TIL.

Validation of gene expression

RNA from immunopurified TEC, cultured HUVEC or whole tumor specimens was

isolated using TRIzol. Real-time quantitative RT-PCR was performed as detailed

elsewhere1. Primers are provided in Supplemental Table 1. All transcripts were confirmed

using 3% agarose gel electrophoresis. In all cases, expression was normalized to � -actin.

Protein was extracted from homogenized whole tumor specimens in RIPA buffer. Western

blot was performed as previously described2 using anti-ETBR (Santa Cruz Biotechnology);

anti� � -actin (AC-15, SIGMA); and secondary HRP-conjugated anti-rabbit antibody.

Immunohistochemistry was performed using mouse anti-ETBR (clone 1922-225, Abcam)

and Vectastain kit (Vector).

In vitro experiments

HUVEC grown at 30-40% confluence in Biocoat 96-well plates in EGM2 media

(Cambrex) were treated with combinations of recombinant human ET-1 (10 nM, Anaspec);

BQ-788 (200 nM, American Peptide); L-NAME (5 mM, SIGMA); DETANO (100 � M,

SIGMA) for 24h, and then TNF-� for 4h (20 ng/ml, SIGMA). Control cells were kept in

EGM2 media; or treated with TNF-� for 4h. Cells were rinsed twice; supplemented with

fresh EGM2 media; and T cells were added to the plates. Peripheral blood lymphocytes

from normal donors were obtained through elutriation and stimulated for 48h with anti-

CD3/CD28 beads in RPMI-1640. Greater than 97% pure T cells were labeled with CFSE;

washed; let adhere to pre-treated HUVEC for 1h; and washed 5 times with PBS.

Fluorescence was detected in 96-wells using Packard Fluorochrome. In some experiments,

HUVEC were not treated with TNF-� . In some experiments, anti-ICAM-1 neutralizing

antibody (LB-2, 20 � g/ml, BD Bioscience) was added to HUVEC 1h before the T cells. To

measure adhesion molecules and chemokines at the mRNA level in HUVEC treated under

similar experimental conditions, cells were plated in 6-well plates; collected at 24h; and

RNA was isolated using TRIzol. To measure ICAM-1 protein expression by

immunofluorescence under similar experimental conditions as above, cells were plated in 8-

well chamber slides (Lab-TekII, Nalgene) and stained using monoclonal antibody against

human ICAM-1 (LB-2, 1:100, BD Bioscience). Plates were inspected under an

epifluorescent microscope (E800 Nikon) at 40X and 100X magnification. For statistical

analysis, six 40X fields were counted.

Immune response analyses

ELISPOT analysis was performed as previously described3. Briefly, splenocytes were

plated in duplicate in 96-wells MAIP plates (Millipore) at a density of 2.5x105 and 5x105

cells/well. Cells were incubated for 20h at 37oC with 1 � g/ml suspension of E7

RAHYNIVTF peptide. Concanavalin A was used as positive internal control for each

mouse at 5 � g/ml. Plates were washed and incubated for 12hrs at 4oC with 50 � l/well of

biotin-conjugated rat anti-mouse IFN-� . After extensive washing, 50 � l/well of NBT/B-CIP

(Pierce) were added until development of spots was clearly visible. Plates were then

washed; air dried; and spots counted using an automated ELISPOT reader. Intracellular

cytokine staining was performed as previously described3. Briefly, 1-2x106 mouse

splenocytes in 1 ml RPMI 10% FCS were incubated with E7 RAHYNIVTF peptide (5-6

� g/ml, EZBiolab) and brefeldin A (1 � g/ml; BD Pharmingen) at 37°C for 12-16 hours.

Cells were washed; stained with surface antibodies; fixed, permeabilized; and incubated

with the IFN-� -FITC antibody (BD Pharmingen). Cells were fixed with 1% formaldehyde

solution in PBS and analyzed on a FACS-Canto flow cytometer, using Diva software

(Becton Dickinson).

Statistical analyses

Descriptive statistics were performed with SPSS♦. The Kaplan-Meier method was used to

estimate all survival curves from human and animal studies. The log-rank statistic was used

to compare the progression-free and overall survival distributions of patients with tumors

exhibiting low ETBR expression to those with high expression using SAS 9.121. Hazard

ratios for ETBR expression were obtained from Cox proportional hazard models and

presented with their 95% confidence intervals. The Wilcoxon rank-sum test was used to

compare ETBR expression between groups defined by intraepithelial TIL. In vitro

experiments and in vivo tumor volumes were analyzed using ANOVA and Student’s t-test.

Mouse survival curves were compared with the log-rank statistic.

References

1. Buckanovich, R.J., et al. Tumor vascular proteins as biomarkers in ovarian cancer.

J Clin Oncol 25, 852-861 (2007).

2. Conejo-Garcia, J.R., et al. Tumor-infiltrating dendritic cell precursors recruited by a

beta-defensin contribute to vasculogenesis under the influence of Vegf-A. Nature

medicine 10, 950-958 (2004).

3. Facciabene, A., et al. DNA and adenoviral vectors encoding carcinoembryonic

antigen fused to immunoenhancing sequences augment antigen-specific immune

response and confer tumor protection. Human gene therapy 17, 81-92 (2006).