SUPPLEMENTARY MATERIAL AND METHODS
Generation of mouse DC vaccines: Antitumor DC vaccines were generated as previously described [18]. Briefly, bone marrow precursors were obtained from BALB/c mice and cultured in RPMI medium (Gibco, Waltham, MA) containing 10 ng/ml IL-4 and 10 ng/ml GM-CSF
(BioLegend, San Diego, CA) [18]. Non-adherent cells were harvested 7 days later with a yield of
0.6-2.5x106 cells per mouse and a purity of over 90%. DCs were incubated with tumor lysates (1
DC: 3 tumor cells) from either tumor cell line for 3 h at 37ºC. Then, 3-5x105 DCs were washed and activated with 10 µg/ml CpG1826 and immediately injected s.c. into the mouse flank.
Breast cancer cell lines
Murine breast cancer cells: The LM3 cell line, developed from a murine mammary adenocarcinoma that spontaneously arose in a BALB/c mouse at the animal care facility of the
Instituto de Oncología “Angel H. Roffo” (Buenos Aires, Argentina) [24] was cultured in MEM with
10% FBS, 1% L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin. 4T1 cells, a highly metastatic breast cancer cell line derived from a spontaneously arising BALB/c mammary tumor
(ATCC, Cat# CRL-2539, kindly provided by Dr Osvaldo Podhajcer from Fundación Instituto
Leloir, Buenos Aires, Argentina) were cultured in RPMI 1640 medium containing 10 % FBS
(Natocor, Cordoba, Argentina), 1 % L-glutamine (Life Technologies, MA), 100 IU/ml penicillin
(Life Technologies, MA) and 100 µg/ml streptomycin (Life Technologies, MA). To generate tumor cell lysates, LM3 or 4T1 tumor cell suspensions were subjected to four freeze-thaw cycles, centrifuged at 1000 rpm for 5 minutes, and the supernatant was kept at -70ºC until the moment of DC vaccine preparation.
Human breast cancer cells: Human MCF7 breast cancer cells (kindly provided by Dr Paula
Maloberti, Instituto de Investigaciones Biomédicas, Facultad de Medicina, Universidad de
Buenos Aires) were cultured in DMEM high glucose (EMEVE, Buenos Aires, Argentina) containing 10 % FBS, 100 IU/ml penicillin and 100 µg/ml streptomycin (Life Technologies, MA). Human MDA-MB-231 breast cancer cells were cultured in supplemented RPMI 1640 medium and MCF10 cells, a non-tumorigenic breast epithelial line, were cultured in DMEM/F12 (EMEVE,
Buenos Aires, Argentina), containing 1% L-glutamine, 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 5 ng/ml insulin, 500 ng/ml Hydrocortisone and 5% FBS. MDA-MB-231 and
MCF10 cells were kindly provided by Dr. Andrea Randi (Departamento de Bioquímica Humana,
Facultad de Medicina, Universidad de Buenos Aires).
Flow cytometry
Mononuclear cells were purified from the spleen and tumors as previously described [18,23].
Briefly, spleens were removed and smashed to release splenocytes. Red blood cells were lysed by incubating the preparation in 3 volumes of ice cold ammonium-chloride-potassium solution
(ACK, 0.15 M NH4Cl, 10 mM KHC03, 0.1 mM Na2EDTA) for 3 min and then washed with supplemented RPMI. In order to purify tumor infiltrating immune cells, tumors were dissected and disaggregated with a glass homogenizer. Mononuclear cells were obtained from the interphase of 30-90% Percoll gradient as previously described [23].
Mononuclear cells were washed with flow cytometry buffer (1% FBS) and incubated with anti-
CD45, anti-CD8 and anti-CD4 for 1 h (Biolegend, San Diego, CA, Cat #103105, 100705,
100539, respectively, San Diego, CA). After washing, cells were fixed with fixation/permeabilization buffer according to manufacturer instructions (eBioscience, MA Cat #
00-5523-00). Then, cells were stained with anti-Foxp3 antibody (eBioscience, MA, Cat# 17-
5773-82), washed and maintained in 0.1% sodium azide in PBS at 4°C until analyzed by flow cytometry.
Tumor cells growing in monolayer were harvested with trypsin-EDTA. Cell suspensions were washed and fixed with fixation/permeabilization buffer and Foxp3 staining was performed as described above. Data were analyzed using WinMDI 2.9 software.
Immunocytochemistry Tumor cells were fixed with acetone for 20 min on ice. After washing with PBS, blocking was performed in 2% bovine serum albumin, 10% goat serum in PBS for 2 h and cells were incubated overnight with anti-murine/human Foxp3 antibody (BioLegend, San Diego, CA, Cat#
623802). Then, cells were incubated with FITC-conjugated anti-rabbit IgG secondary antibody
(Vector, Burlingame, CA, Cat# FI-1000) for 1 h. After washed with PBS, cells were incubated with DAPI for 10 min, washed and mounted using Vectashield (Vector, Burlingame, CA,).
Immunofluorescence signal intensity per cell was measured using ImageJ software.
PFA-fixed LM3 and 4T1 tumors were dissected and 30 µm cryostate sections were made.
Immunohistochemistry was performed using free-floating technique in which tumors sections were incubated in liquid throughout the process. Antigen retrieval was performed in 10 mM citrate buffer (pH 6) for 5 min in microwave. Sections were washed with PBS and incubated with
5% Triton TBS buffer for 5 min. Sections were blocked in 2% Triton 10% donkey serum TBS for
1 h. Then, sections were incubated overnight with specific antibodies against mouse CD45,
CD8, and MHCII (Serotec, Cat # MCA-1031GA; MCA-1767GA, MCA-46GA, respectively).
Sections were washed for 90 min and incubated with FITC- or Alexa594-conjugated donkey anti-rat secondary antibodies (Chemicon and Vector, respectively) for 2 h. Sections were mounted in anti-fade mounting medium Vectashield (DAKO).
Western blot
Foxp3 tumor content was analyzed by Western blot in immunecompromised mice on the last day of P60 administration. Proteins from homogenized tumors were extracted in fresh lysis buffer (pH 7.9) containing 10 mM KCl, 2 mM MgCl2, 0.5 mM dithiothreitol (DTT), 1% IGEPAL
CA-630, 0.2 mM ethylenediaminetetraacetic acid (EDTA) in 10 mM Hepes, 2.5 mM sodium floride, 0.5 mM sodium orthovanadate and protease inhibitor cocktail (Sigma-Aldrich, St. Louis,
MO, Cat# P8340). Following centrifugation at 12000 g for 30 min, the supernatant was recovered and protein concentration of each sample was determined by the Bradford protein assay (Bio-Rad, Hercules, CA). 10-20 µg of protein were size-fractionated in 12% sodium dodecylsulphate-polyacrylamide gel (Bio-Rad, Cat#162-0177) and electrotransferred to polyvinyl difluoride (PVDF) membranes. Blots were blocked for 2 h in 5% bovine serum albumin
(BSA)-Tris-buffered saline- 0.1% Tween 20 at room temperature and incubated overnight with anti-Foxp3 antibody (eBioscience, Cat# 14-7979-80) and β-actin (Sigma-Aldrich, Cat# A2066).
This was followed by incubation with HRP-conjugated anti-rabbit IgG secondary antibody
(Millipore, Cat# AP103P) for 1 h. Immunoreactivity was detected by enhanced chemiluminescence (ECL plus, Amersham Biosciences). Blots were scanned with an Image
Quant 300 imager and analyzed by Image Quant TL software (Amersham Biosciences, GE
Healthcare). Foxp3 intensity was normalized with the corresponding β-actin blot.
Viability assessment (MTT)
5x104 tumor cells were cultured in 96-well plates and incubated in the presence of 50 µM P60 or
P301 for 24 h. Then, cells were washed with Krebs solution (0.9% NaCl, 1.15% KCl, 1.22%
CaCl 2, 2.1% KH2PO 4, 3.8% MgSO 4, 2.6% NaHCO 3) and incubated with 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazol (MTT, Molecular Probes) for 4 h. The reaction was then quenched using 6N HCl solution. Absorbance was determined in 96-well plate spectrophotometer (Spectramax Plus, Molecular Devices) at 595 nm.
Proliferation Assay
5x104 tumor cells were cultured in 96-well plates and incubated with 50 µM P60 or P301 for 24 h. Then, BrdU labeling solution (1:100) was added to the media for additional 4 h and BrdU incorporation into cellular DNA strands was assessed by ELISA following manufacturer’s instructions (ROCHE, Cat# 11647229001). Absorbance was measured in 96-well plate spectrophotometer at 450 nm.
IL-10 ELISA
IL-10 was measured in supernatants of 1x105 tumor cells cultured for 24 h in the presence of 50
µM P60 or P301 by ELISA following the manufacturer’s protocol (BioLegend Mouse IL-10 ELISA MAX™ Deluxe, Cat#4314). Absorbance was read in 96-well plate spectrophotometer at
450 nm.