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www.sciencemag.org/cgi/content/full/334/6062/1573/DC1 Supporting Online Material for Autophagy-Dependent Anticancer Immune Responses Induced by Chemotherapeutic Agents in Mice Mickaël Michaud, Isabelle Martins, Abdul Qader Sukkurwala, Sandy Adjemian, Yuting Ma, Patrizia Pellegatti, Shensi Shen, Oliver Kepp, Marie Scoazec, Grégoire Mignot, Santiago Rello-Varona, Maximilien Tailler, Laurie Menger, Erika Vacchelli, Lorenzo Galluzzi, François Ghiringhelli, Francesco di Virgilio, Laurence Zitvogel, * Guido Kroemer * *To whom correspondence should be addressed. E-mail: [email protected] (G.K.); [email protected] (L.Z.) Published 16 December 2011, Science 334, 1573 (2011) DOI: 10.1126/science.1208347 This PDF file includes: Materials and Methods SOM Text Figs. S1 to S16 References (30–73)

Transcript of Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v...

Page 1: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

www.sciencemag.org/cgi/content/full/334/6062/1573/DC1

Supporting Online Material for

Autophagy-Dependent Anticancer Immune Responses Induced by Chemotherapeutic Agents in Mice

Mickaël Michaud, Isabelle Martins, Abdul Qader Sukkurwala, Sandy Adjemian, Yuting

Ma, Patrizia Pellegatti, Shensi Shen, Oliver Kepp, Marie Scoazec, Grégoire Mignot, Santiago Rello-Varona, Maximilien Tailler, Laurie Menger, Erika Vacchelli, Lorenzo

Galluzzi, François Ghiringhelli, Francesco di Virgilio, Laurence Zitvogel,* Guido Kroemer*

*To whom correspondence should be addressed. E-mail: [email protected] (G.K.);

[email protected] (L.Z.)

Published 16 December 2011, Science 334, 1573 (2011)

DOI: 10.1126/science.1208347

This PDF file includes:

Materials and Methods SOM Text Figs. S1 to S16 References (30–73)

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Material and Methods

Chemicals, cell lines and culture conditions

Unless otherwise indicated, media, antibiotics and supplements for cell culture were purchased

from Gibco-Invitrogen (Carlsbad, CA, USA), plasticware from Corning B.V. Life Sciences

(Schiphol-Rijk, The Netherlands), and chemicals from Sigma-Aldrich (St Louis, MO, USA).

Bafilomycin A1 was purchased from Tocris (Bristol, United Kingdom), carbobenzoxy-valyl-

alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk) from Bachem (Bubendorf,

Switzerland) and NGXT-191 (NGXT) from Chembridge (San Diego, CA, USA). G418 sulfate

was purchased from Calbiochem (San Diego, CA, USA), while puromycin and zeocin were

obtained from Invivogen (San Diego, CA, USA).

Murine colon carcinoma CT26 cells (class I MHC haplotype H-2d, syngenic to BALB/c mice),

murine fibrosarcoma MCA205 cells (class I MHC haplotype H-2b, syngenic for C57BL/6 mice)

cells and their derivatives were cultured in RPMI 1640 medium. Human osteosarcoma U2OS

cells and derivatives were maintained in DMEM, and mouse embryonic fibroblasts (MEFs) in

DMEM plus 1% nonessential amino acids (NEAA). All media were supplemented with 10%

heat-inactivated fetal bovine serum, 10 mM HEPES buffer, 10 U/mL penicillin sodium and 10

μg/mL streptomycin sulfate.

U2OS cells stably co-expressing a green fluorescent protein (GFP)-LC3 fusion or a high mobility

group box 1 (HMGB1)-GFP chimera were generated as previously described (30) and maintained

under 200 µg/mL G418 selection pressure.

To obtain autophagy-deficient CT26 and MCA205 cells, a set of plasmids encoding short-hairpin

RNAs (shRNAs) specific for murine Atg5 and Atg7 plus a control shRNA were obtained from

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Origen (Rockville, MD, USA). These plasmids were used to generate control cells (SCR) as well

as cells stably depleted of Atg5 (Atg5KD) and Atg7 (Atg7KD). Atg5 and Atg7 knockdown was

evaluated in single clones by immunoblotting. SCR, Atg5KD or Atg7KD CT26 and MCA205 cells

were maintained under 10 or 5 µg/mL puromycin selection, respectively.

To obtain the stable expression of a GFP-LC3 fusion, the corresponding construct was

transfected into CT26 and U2OS cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA,

USA), following the manufacturer’s instructions. The GFP-LC3-encoding plasmid was obtained

from Addgene (31, 32). Transfected cells were maintained under 500 µg/mL G418 selection, and

GFP-expressing clones were selected by cytofluorometry before the experiments.

To generate cells stably expressing the firefly luciferase from Photinus Pyralis on the plasma

membrane (pmeLUC), SCR, Atg5KD or Atg7KD CT26 cells were co-transfected with a pmeLUC-

encoding construct and the pcDNA3 plasmid (33) by using Lipofectamine 2000, as indicated by

the manufacturer. Two days later, 1 mg/mL G418 was added to select transfected clones. The

amount of G418 was reduced to 0.2 mg/mL 20 days after transfection, and stable pmeLUC-

expressing clones were maintained under continuous selection.

To generate cells stably expressing murine CD39, the corresponding gene (ENtpd1) was cloned

into the pENTR/D-TOPO vector (Invitrogen), according to the manufacturer's instruction, with

the following primers: sense: 5' CACCATGGAAGATATAAAG 3'; anti-sense: 5'

TACTGCCTCTTTCCAGAA 3'. To insert the gene into a lentiviral vector, pENTR/D-TOPO

vector containing ENtpd1 was recombined with pLenti6/V5-DEST (Invitrogen), following the

manufacturer's recommendations. The identity of this construct was confirmed by restriction with

BsrGI (Promega, Madison, WI, USA) and sequencing (Millegene, Labege, France). ENtpd1-

encoding viruses were produced using the ViraPower Lentiviral expression system (Invitrogen)

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and used to infect MCA205 cells. Cells expressing CD39 were selected in 10 µg/mL blasticidine

and characterized by surface immunofluorescence (see below).

RNA interference

A control small-interfering RNA (UNR, Universal negative control #1) as well as siRNAs

specific for murine Atg3 (sense 5’-CAUAUCUUCCGACAGACAdTdT-3’), murine Atg5 (sense

5’-CAUCAACCGGAAACUCAUdTdT-3’), murine Atg7 (sense 5’-

AGUUUCCAGUCCGUUGAAdTdT-3’), murine Atg10 (sense 5’-

CUAAAGAAUUCACAGAAAdTdT-3’), human ATG5 (sense 5’-

CCUUUGGCCUAAGAAGAAATTdTdT-3’), human ATG7 (sense 5’-

CGACUUGUUCCUUACGGAATTdTdT-3’) and human BCN1 (sense 5’-

CAGUGGAUCUAAAUCUCAATTdTdT-3’) were purchased from Sigma-Proligo (The

Woodlands, TX, USA). A pre-designed commercial siRNA specific for murine Beclin-1 (BCN1,

sc-29798) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

CT26, MCA205 and U2OS cells were transfected (at 30-40 % confluence) with the HiPerFect

transfection reagent (Qiagen, Hilden, Germany) - previously complexed with 100 nM siRNA - as

instructed by the manufacturer. Transfected cells were used for experiments no earlier than 48 h

after transfection. Protein knockdown was confirmed by immunoblotting.

Immunoblotting

For immunoblotting, approximately 1 x 106 cells were washed with cold PBS and lysed following

standard procedures (34). Forty µg of proteins were separated according to molecular weight on

NuPAGE® Novex® Bis-Tris 4–12% pre-cast gels (Invitrogen) and electrotransferred to

nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Unspecific binding sites were blocked

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by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5%

non-fat powdered milk or bovine serum albumin, followed by overnight incubation at 4 °C with

primary antibodies specific for the following proteins: β-actin (MAB1501, Millipore, Temecula,

CA, USA); ATG5 (A0731, Sigma-Aldrich), ATG7 (A2856, Sigma-Aldrich), BCN1 (SC-11427,

Santa-Cruz Biotechnology), or LC3B (#2775, Cell Signaling Technology, Danvers, MA, USA).

Primary antibodies were detected with the appropriate horseradish peroxidase-labeled secondary

antibodies (Southern Biotechnologies Associates, Birmingham, UK) and were revealed with the

SuperSignal West Pico chemoluminescent substrate (Thermo Fisher Scientific, Rockford, IL,

USA) or ECL Plus Western Blotting Detection System (GE Healthcare, Little Chalfont, UK). The

abundance of β-actin was monitored to ensure equal loading.

Immunofluorescence

For the detection of surface-exposed CRT, trypsinized cells were fixed with 0.25%

paraformaldehyde in PBS (5 min, 4°C), washed in PBS and subjected to indirect

immunofluorescence with a rabbit anti-CRT antibody (Abcam) revealed with an appropriate

secondary Alexa Fluor® 488 conjugate. CD39 surface expression was monitored upon staining

with CD39-antibody (Biolegend, San Diego, CA, USA) revealed by means of an appropriate

Alexa Fluor® 488 conjugate (Invitrogen). Finally, cells were subjected to cytofluorometric

analysis (FACScan from Becton Dickinson, Mountain View, CA, USA) in the presence of 5

µg/mL propidium iodide (PI). Analysis was restricted to living (PI-) cells.

Cell death assays

5 x 105 CT26 or MCA205 cells were treated with the indicated agents at the indicated

concentration for 18 h followed by the cytofluorometric assessment of PI uptake and

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phosphatidylserine (PS) externalization (35). To this aim, cells were washed twice with PBS and

resuspended in 1X binding buffer supplemented with FITC-conjugated AnnexinV (BD

Biosciences, San Diego, CA, USA) and 0,5 μg/mL PI, following the manufacturer’s instructions.

All cytofluorometric determinations were carried out using a FACSCalibur or a FACScan

cytofluorometer (Becton Dickinson) equipped with a 70 µm nozzle. First line statistical analysis

of cytofluorometric results was performed by using the CellQuest™ software (BD Biosciences),

by gating on the events characterized by normal forward scatter and side scatter parameters.

ATP release assays in vitro

Extracellular and intracellular ATP levels were measured by the luciferin-based ENLITEN® ATP

Assay (Promega) and ATP assay (Calbiochem, Nottingham, UK) kits, respectively, in excess of

luciferin and luciferase, as indicated by the manufacturer. ATP-driven chemoluminescence was

recorded on a Fluostar multiwell plate luminometer (BMG Labtech, Offenburg, Germany).

Automated high content microscopy and videomicroscopy

Seven x 103 U2OS cells expressing GFP-LC3 were transfected with the indicated siRNAs and

seeded in 96-well Black/Clear Imaging Plates (BD Biosciences) that had been previously coated

with poly-L-lysine. Two days later, cells were treated with the indicated drugs for 6 h or 48 h,

respectively, and subsequently fixed in 4% paraformaldehyde (w/v in PBS) supplemented with

10 μM Hoechst 33342 for 20 min. After one wash with PBS, four view fields per well were

acquired by means of an automated microscope (BD Pathway 855, from BD Biosciences

equipped with PhotoFluor II light sources from 89 North, Burlington, VT, USA). Images were

segmented and analyzed for the quantification of cytoplasmic (non-nuclear) GFP-LC3 puncta, by

using the BD AttoVision software version 1.6 (BD Biosciences).

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Similarly, CT26 or MCA205 cells were seeded in 96-well Black/Clear Imaging Plates that had

been previously coated with poly-L-lysine. One day later, cells were treated with the indicated

drugs for 24 h, fixed in 4% paraformaldehyde, washed and then subjected to indirect

immunofluorescence detection of LC3B upon staining with a LC3B-specific antibody (M152-3

from Abcam). Revelation was performed by means of an appropriate Alexa Fluor® 488 conjugate

(Invitrogen). Nuclear counterstaining was achieved upon incubation with 10 µM Hoechst 33342.

Finally, nine view fields per well were acquired by means of the BD Pathway 855 microscope

and analyzed for cytoplasmic LC3 puncta by means of the BD AttoVision software.

Detection of HMGB1 release

2.5 x 105 MCA205 cells were seeded in 6-well plates, let adhere overnight and then treated with

the indicated drugs for further 24 h. Supernatants were collected and cleared from dying tumor

cells by centrifugation (800 g, 5 min), then frozen at -80 °C or immediately analyzed for HMGB1

abundance. HMGB1 quantification was performed by means of an enzyme-linked

immunosorbent assay (ELISA) kit (HMBG1 ELISA kit II, Gentaur Europe, Kampenhout,

Belgium), according to the manufacturer's instructions.

Targeted analysis of extracellular ATP by HPLC-QQQ

Two 2 x 105 SCR, Atg5KD or Atg7KD CT26 cells were seeded in 6-well plates, let adapt overnight

and treated with 1 μM MTX (or an equivalent volume of PBS) for 15 h. Culture supernatants

were discarded, cells were washed with PBS and incubated for 50 min in 0.8 mL HBSS.

Thereafter, HBSS was collected and cleared from cellular debris by centrifugation (5 min, 800 g).

Finally, HBSS samples were stored at -80°C until HPLC-QQQ analysis. ATP quantification was

performed on a Rapid Resolution Liquid Chromatography (RRLC) 1200SL system coupled to a

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6410Triple Quadripole (QQQ) mass spectrometer, both from Agilent (Agilent Technologies,

Santa Clara, CA, USA).

RRLC analysis was done on 150 x 2.1 mm, 3.5 μM Eclipse Plus columns (Agilent) with water

containing 4 mM dimethylhexylamine (DMHA) and 0,01% acetic acid in channel A and

acetonitrile in channel B, in gradient mode: t = 0 min 2% B; t = 9 min 32% B; t = 10 min 95% B;

T = 12 min 95% B, re-equilibration time of 6 min. Mass spectrometry was performed in positive

electrospray ionization mode at + 4kV on the QQQ system operating in MRM mode. MRM

transitions were optimized with ATP standards with direct infusion, and two transitions were

recorded. Parent ions were adduct ions of DMHA more sensitive than proton adducts:

ATP 637.2 > 508 CE (collision energy)12 V; fragmentor 200V/ 637.2 > 136 CE 44V.Data

collection and analysis were performed with the MassHunter software (Agilent).

Animal experimentations

Animals were maintained in specific pathogen-free conditions, and experiments followed the

Federation of European Laboratory Animal Science Association (FELASA) guidelines. Animal

experiments were approved by the local Ethics Committee (CEEA IRCIV / IGR n°26, registered

with the French Ministry of Research) and were in compliance with EU 63/2010 directive.

Animals were used between 6 and 20 weeks of age and those bearing tumors exceeding 20–25%

body mass were euthanatized. BALB/c (H-2d), C57BL/6 (H-2b) mice were obtained from

Janvier (Le Genest St Isle, France) or Harlan (Gannat, France). nu/nu and Rag γ mice were from

the internal animal facility (IGR, Villejuif, France). C57BL/6J mice expressing GFP under the

control of a dendritic cell-specific promoter (CD11c-DTR-GFP) were obtained from The

European Mouse Mutant Archive (Munich, Germany) and bred in the IGR animal facility.

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Real time in vivo imaging of ATP release

BALB/c mice were inoculated with 5 x 105 CT26 clones stably expressing pmeLUC, and tumor

growth was monitored every 2 days by means of a common caliper. When tumors became

palpable (about 2 weeks after injection), animals were divided in three experimental groups. The

first one was injected with 200 µl PBS only (control group). The second one was

intraperitoneally injected with 5.17 mg/kg MTX in 200 µl of PBS. The third one was

intraperitoneally injected with 5.17 mg/kg MTX in 200 µl of PBS and subjected to the

intratumoral inoculation of 1.6 mg/kg ARL67156 in 100 µl PBS. Thereafter, mice were

anesthetized and imaged with a high sensitivity CCD IVIS® Lumina camera mounted on a dark

imaging chamber (Caliper, Hopkinton, MA, USA). D-luciferin (150 mg/kg) was intraperitoneally

administered to mice 15 minutes before acquisitions, which were performed immediately before

and 48 h after injections. The regions of interest were identified around tumor sites and in vivo

luminescence was quantified as photons/s using the Living ImageH software (Caliper).

Anti-tumor vaccination experiments

CT26 cells were treated by 2 μM MTX (alone, or in combination with 20 μM

hydroxychloroquine or 50 nM bafilomycin A1) or subjected to UV irradiation (1.5 kJ/m2), and 24

h later were subcutaneously inoculated (3 x 106 in 200 μl PBS, pH 7.4) into the lower flank of 6-

week-old female BALB/c mice. In these experiments, aliquots of cells were subjected to

AnnexinV/PI co-staining to ensure that 70±10% of the cells were dead (Annexin V+PI+) or dying

(AnnexinV+PI-) at the moment of inoculation. Seven days later, 5 x 105 living CT26 cells (in 200

µL PBS, pH 7.4) were inoculated - alone or in combination with 0.5 x 10-3 mg/kg interleukin 1-β

(IL1-β), 1.6 mg/kg ARL67156, 100 mg/kg suramin, or 4 mg/kg periodate oxidized sodium salt

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(oxy-ATP) - into the controlateral flank, as previously described (1). Mice were then monitored

for the appearance of tumors three times a week for 60 days.

Similarly, MCA205 cells were incubated with 1 µM MTX alone or in combination with 20 µM

hydroxychloroquine, 50 nM bafilomycin A1 for 24 h and then subcutaneously inoculated (3 x 105

in 200 μl PBS, pH 7.4) - alone or in the presence of IL1-β, ARL67156 or oxi-ATP, as above -

into the lower flank of 6-week-old C57BL/6 female mice. Seven days later, 3 x 104 untreated

MCA205 cells were inoculated into the controlateral flank and the appearance of tumors was

checked three times a week for 60 days.

Chemotherapy of established tumors in mice

BALB/c and nu/nu mice were subcutaneously inoculated with 5 x 105 SCR, Atg5KD or Atg7KD

CT26 cells. Similarly C57BL/6 and nu/nu mice were inoculated with 2 × 105 SCR, Atg5KD or

Atg7KD MCA205 cells or, alternatively, with MCA205 cells engineered to express CD39 on their

surface or transfected with a control construct. When tumor size reached 40–80 mm2, mice were

assigned into homogenous groups (5 mice per group), then treated either intraperitoneally or

intratumorally with 5.17 mg/kg or 1.7 mg/kg MTX (in 200 μl sterile water), respectively, or

intraperitoneally with 10 mg/kg oxaliplatin, alone or in combination with 1.6 mg/kg intratumoral

ARL-67156 (in 100 μl PBS, pH 7.4) or 12.5 mg/kg intratumoral NGXT-191 (in 100 μl PBS,

pH7.4 + 2.5% DMSO). Tumor growth was then monitored with a common caliper every 2-3 days

for up to 25 days. When appropriate, ARL-67156 and NGXT-191 were re-injected three days

after MTX administration.

Cytofluorometric quantification of tumor-infiltrating lymphocytes

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BALB/c mice were subcutaneously inoculated with 5x 105 SCR or Atg5KD CT26 cells, then

treated by intraperitoneal MTX and intratumoral ARL67156, as described above. Nine days after

treatment, tumor masses were carefully removed, cut into small pieces with scissors and

incubated for 30 min at 37° C in digesting buffer (400 U/mL collagenase IV plus 150 U/mL

DNase I in RPMI1640). Single cell suspensions were obtained by grinding digested tissues with

syringe plungers and filtering through a 70 µm cell strainer. After one wash in RPMI1640,

tumor-infiltrating leukocytes were enriched by density separation on Lympholyte®-M (Cedarlane

Labs, Burlington, Canada) and resuspended at 1×107 cells/mL in PBS. Unspecific interactions

were blocked with 10 µg/mL anti-CD16/CD32 (5 min, +4° C). Cells were then stained with

CD45.2-PerCPCy5.5 (eBioscience, San Diego, CA, USA), CD3-FITC (17A2) (BioLegend),

CD4-Pacific Blue (RM4-5) (BD Biosciences), CD8-APC-cy7 (53.6.7) (BioLegend) and TCR δ–

APC (GL-3) (eBioscience),washed with PBS and stained with IL-17A-PE (TC11-18H10) (BD

Biosciences), IFN-γ PE-Cy7 (XMG1.2) (BioLegend) by following the Cytofix/Cytoperm™ kit’s

(BD Biosciences) instructions. The LIVE/DEAD Fixable Dead Cell Stain kit (Molecular Probes-

Invitrogen) was used to discriminate between living and dead cells.

In situ immunofluorescence

CD11c-DTR-GFP C57BL/6 mice were subcutaneously inoculated with 5 x 105 SCR, Atg5KD or

Atg7KD MCA205 cells, allowed to develop palpable tumors and then treated with 5 mg/kg

intraperitoneal MTX (in 200 μl sterile water), alone or in combination with 1.6 mg/kg

intratumoral ARL-67156 (in 100 μl PBS, pH 7.4). Forty-eight hours later, tumor masses were

collected and fixed for 4 h in neutral buffered formalin at room temperature, incubated overnight

in 30% saccharose (w/v in sterile water), and then embedded in Tissue-TekTM OCT compound

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(Sakura, Villeneuve d’Ascq, France) and snap-frozen on dry ice. Five μm-thick cryosections

were prepared and immunohistochemistry was performed following standard procedures (5).

Sections were rinsed with PBS and unspecific binding sites were blocked by incubation in 10%

fetal bovine serum (FBS) (v/v in PBS) for 45 min. Thereafter, sections were incubated with an

anti-cleaved caspase-3 antibody (#9661, Cell Signalling) in 10% FBS for 1h, rinsed again, and

incubated with appropriate secondary antibodies (in 10% FBS) for 30 min. Finally, slides were

washed and incubated for 5 min in 10 μM Hoechst 33342, washed once more and mounted with

Fluoromount-G™ (Southern Biotechnologies) . For each tumor, 10 view fields showing caspase-

3 activation were acquired with a Leica SPE confocal microscope. Pictures were analyzed with

the open-source software Image J (freely available at rsbweb.nih.gov/ij/) to determine the

percentage of CD11c+ cells and of cells exhibiting caspase-3 activation. In another series of

experiments, HMGB1 was detected upon staining with a polyclonal rabbit antiserum (Abcam)

revealed by an anti-rabbit IgG Alexa Fluor® 488 conjugate (Invitrogen).

Immunohistochemestry

C57BL/6 mice were subcutaneously inoculated with 5 x 105 SCR, Atg5KD or Atg7KD MCA205

cells, allowed to develop palpable tumors and then treated with 5 mg/kg intraperitoneal MTX (in

200 μl sterile water). Forty-eight hours later, tumor masses were collected and fixed for 4 h in

neutral buffered formalin at room temperature, incubated overnight in 30% saccharose (w/v in

sterile water). Tumors were then embedded in Tissue-TekTM OCT compound (Sakura,

Villeneuve d’Ascq, France) and snap-frozen on dry ice. Five μm-thick cryosections were

prepared, and immunohistochemistry was performed following standard procedures. The

NOVOLINK Polymer Detection System from Menarini diagnostics (Rungis, Fance) and an LC3-

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specific antibody (5F10, from Nanotools, Teningen, Germany), were used according to the

manufacturer's instructions.

Priming assays

Five 5 x 105 SCR, Atg5KD or Atg7KD CT26 cells or 2 x 105 SCR, Atg5KD or Atg7KD MCA205

cells were injected into the footpad of BALB/c or C57BL/6 mice, respectively. Six days later,

popliteal lymph nodes cells were collected and re-stimulated in vitro (for 72 h) with heat-killed (5

min at 42°C, followed by 1 cycle of freezing/thawing in liquid nitrogen) 3 x 104 CT26 or

MCA205 cells, followed by the assessment of interferon-γ secretion by using an ELISA kit (BD

Biosciences).

Statistical analyses

Data were compared by the Kaplan Meyer, χ2, or Student’s t tests, as appropriate. All p values

were two tailed. p values < 0.05 were considered as statistically significant for all experiments.

Data were treated with Prism 5 (GraphPad Software, La Jolla, CA, USA) and Excel 2007

(Microsoft, Rockville, MD, USA).

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Supplementary References

30. I. Martins et al., Restoration of the immunogenicity of cisplatin-induced cancer cell death

by endoplasmic reticulum stress. Oncogene 30, 1147-1158 (2010).

31. S. Kimura, T. Noda, T. Yoshimori, Dissection of the autophagosome maturation process by

a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3, 452-460 (2007).

32. I. H. Lee et al., A role for the NAD-dependent deacetylase Sirt1 in the regulation of

autophagy. Proc Natl Acad Sci U S A 105, 3374-3379 (2008).

33. P. Pellegatti, S. Falzoni, P. Pinton, R. Rizzuto, F. Di Virgilio, A novel recombinant plasma

membrane-targeted luciferase reveals a new pathway for ATP secretion. Mol Biol Cell 16,

3659-3665 (2005).

34. L. Galluzzi et al., miR-181a and miR-630 regulate cisplatin-induced cancer cell death.

Cancer Res 70, 1793-1803 (2010).

35. L. Galluzzi et al., Methods for the assessment of mitochondrial membrane permeabilization

in apoptosis. Apoptosis 12, 803-813 (2007).

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Supplementary Discussion - The ambiguous role of autophagy in oncogenesis,

tumor progression and therapeutic responses.

The ambiguous role of autophagy in oncogenesis, tumor progression and cancer therapy has been

the subject of multiple recent reviews (23, 36-39).

Numerous reports indicate that a specific set of genetic alterations can inhibit autophagy in tumor

cells and that this may contribute to tumorigenesis. Multiple oncoproteins, including (but not

limited to) phosphatidylinositol 3-kinase (PI3K), AKT1, RAS and anti-apoptotic proteins from

the BCL-2 family, inhibit autophagy (40-44). Along similar lines, several oncosuppressor

proteins stimulate autophagy, meaning that their loss reduces autophagy (21, 45, 46). Among

these are BH3-only proteins, death-associated protein kinase 1 (DAPK1, a kinase that operates at

the interface between cell death and autophagy regulation), phosphatase and tensin homolog

(PTEN, the phosphatase that functionally antagonizes PI3K), tuberous sclerosis proteins 1 and 2

(TSC1 and TSC2, which are mutated in hereditary forms of tuberous sclerosis), p19ARF (an

indirect activator of TP53), ARHI (a maternally imprinted RAS-related oncosuppressor), NF1

(whose loss-of-function mutations lead to the hereditary syndrome known as neurofibromatosis)

as well as LKB1/STK11 (a protein kinase whose activity impinges on the central regulator of

autophagy mammalian target of rapamycin, mTOR) (47-55). Moreover, Beclin-1, which is

required for autophagy induction, acts as a haploinsufficient tumor suppressor protein (56), and

other essential autophagy mediators (such as ATG4C, UVRAG and BIF1) exert bona fide

oncosuppressive functions (57-59). Finally, autophagic defects have been shown to accelerate

genomic instability, in turn promoting tumorigenesis (25, 60). Apparently in contrast with these

results - most of which have been obtained in murine models of tumorigenesis - rather unspecific

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inhibitors of autophagy such as chloroquine and 2-phenylethynesulfonamide (PES) can prevent

tumorigenesis in Myc-induced lymphoma models with intact immunity (61, 62). Nevertheless, it

is not clear whether the oncosuppressive functions of chloroquine and PES truly depend on

autophagy inhibition or whether they reflect an off-target effect. Chloroquine is a lysosomotropic

agent that affects multiple cellular functions, and PES reportedly interacts with chaperones from

the heat-shock protein family, notably HSP70, which are known to contribute to oncogenesis by

favoring the adaptation of cancer cells to intracellular stress conditions (63). Taken together,

these observations suggest that autophagy may act as an oncosuppressor mechanism.

Nonetheless, autophagy plays a complex and sometimes ambiguous role in oncogenesis and

tumor progression. At the cell-autonomous level, indeed, increased autophagy often improves the

fitness of tumor cells and facilitates their survival during tumorigenesis and in response to

anticancer regimens (46). Thus, as tumors progress, cancer cells may upregulate autophagy,

presumably as a mechanism of adaptation to their peculiar metabolic needs in the context of

adverse intracellular (oncogenic stress) and extracellular (nutrient shortage, hypoxia) conditions

(23). This is particularly true for pancreatic cancer, in which tumor cell survival depends on

elevated autophagy (64). Along similar lines, increased levels of autophagy predict poor response

to chemotherapy and shortened survival in human melanoma and perhaps in breast cancer (65-

68). Thus, whereas autophagy appears to inhibit oncogenesis, it operates as a pro-tumor

mechanism during cancer progression and in the response of cancer cells to therapy.

Truly specific pharmacological inhibitors of autophagy are not yet available for preclinical

studies. However, lysosomotropic agents such as chloroquine and 3-hydroxychloroquine (which,

among other effects, prevent the fusion between lysosomes and autophagosomes, thus blocking

the last steps of autophagy) have been used to explore the potential of autophagy inhibition as a

means to sensitize cancer cells to therapy (69). Driven by promising preclinical results and since

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chloroquine and 3-hydroxychloroquine have long been used for the treatment of malaria, they

have rather quickly entered clinical trials (www.clinicaltrials.gov). Among other combinations,

chloroquine is currently being tested in association with radiotherapy for the treatment of

glioblastoma multiforme (NCT00224978) (70) or with tamoxifen for the treatment of breast

cancer (NCT01023477), while 3-hydroxychloroquine is being investigated either as monotherapy

or combined with multiple distinct chemotherapeutic agents in at least 15 different clinical

studies (www.clinicaltrials.gov). This contrasts with reports demonstrating that some

experimental anticancer agents can kill tumor cells by triggering autophagic cell death, i.e., a cell

death subroutine that relies for its execution on the molecular machinery for autophagy (71-73).

Given these complexities and heterogeneities, we insist on the fact that our manuscript does not

constitute a recommendation to induce or suppress autophagy for the treatment of human cancers.

Notwithstanding this caveat, our paper reveals a connection between chemotherapy-induced

autophagy, ATP release and anticancer immune responses in the context of murine tumors. This

may have profound implications for the development of novel strategies that aim at rendering

cancer cell death immunogenic.

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Legends to Supplementary Figures

Supplementary Figure 1. Deficient autophagy but normal calreticulin exposure upon Atg5 or

Atg7 knockdown in CT26 cells. A. Immunoblotting detection of Atg5 and Atg7 in CT26 clones

5217 (transfected with a Atg5-specific shRNA), 717 and 745 (both transfected with a Atg7-specific

shRNA). Clones 5217 and 745 were used in all experiments, and compared to cells expressing a

control, scrambled (SCR) shRNA. B. Representative LC3 distribution in cells that were left

untreated (UNT) or treated for 18h with 1 µM MTX or 75 µM oxaliplatin, as assessed by

immunofluorescence. Quantitative data (cytoplasmic LC3 dots/cell) are reported in Fig. 1B. C.

Representative surface calreticulin (CRT) exposure in cells treated as in B, as assessed by indirect

immunofluorescence and cytofluorometry on intact, living cells that exclude the vital dye

propidium iodide. Quantitative data (percentage of surface CRT+ cells) are shown in Fig. 1B.

Supplementary Figure 2. Requirement for autophagy in the release of ATP by MCA205 cells

responding to chemotherapy in vitro. A. Immunoblotting detection of Atg5 and Atg7 in CT26

clones 5232 (transfected with a Atg5-specific shRNA) and 7141 (transfected with a Atg7-specific

shRNA), as well as in CT26 cell clones expressing expressing a control, scrambled (SCR) shRNA.

B. Representative LC3 distribution in cells that were left untreated (UNT) or treated for 18h with 1

µM MTX or 30 µM oxaliplatin, as assessed by immunofluorescence. C-H. Cells treated as in B

were characterized for LC3 aggregation (upon immunofluorescence and automated image analysis,

C), ATP release in the supernatant (by an enzymatic assay, D), cell surface calreticulin (CRT)

exposure (upon immunofluorescence on intact cells and cytofluorometry E,F), cell death-associated

parameters (upon AnnexinV/PI staining, G) and HMGB1 release in the supernatant (by ELISA, H).

Results are reported as means ± SD of n = 3 independent determinations and asterisks indicate

significant (p<0.05, unpaired Student’s t test) differences as compared to autophagy-competent

(SCR-expressing) cells cultured in similar conditions. 

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Supplementary Figure 3. Deficient autophagy but normal calreticulin exposure upon Atg5 or

Atg7 knockout in mouse embryonic fibroblasts (MEFs). A. Immunoblotting detection of Atg5

and Atg7 in Atg5-/- and Atg7-/- MEFs. Atg5 and Atg7 levels were also monitored in wild-type (WT)

cells as a control condition. B. Immunoblotting detection of LC3 processing in MEFs of the

indicated genotype that were left untreated (UNT) or treated with 250 nM mitoxantrone (MTX),

150 µM oxaliplatin (OXA), or 1 µM rapamycin (RAPA) overnight. C. Representative LC3

distribution in MEFs of the indicated genotype treated as in B, as assessed by immunofluorescence.

Quantitative data are reported in Fig. 1C. D. Representative surface calreticulin (CRT) exposure in

MEFs of the indicated genotype treated as in B, as assessed by indirect immunofluorescence on

intact, living cells. Quantitative data are shown in Fig. 1C.

Supplementary Figure 4. Deficient autophagy but normal calreticulin exposure and HMGB1

release upon siRNA-mediated depletion of ATG5, ATG7, ATG10 or Beclin 1 from human

osteosarcoma U2OS cells. A. Immunoblotting detection of ATG5, ATG7 or Beclin 1 (BCN1) in

U2OS cells transfected with specific siRNAs or with a control siRNA (siUNR). * indicates a non-

specific band B. Representative LC3 distribution in cells stably expressing a GFP-LC3 fusion, upon

transfection with the indicated siRNAs and culture in control conditions (untreated, UNT) or in the

presence of 2 µM mitoxantrone (MTX) for 18 h. Please note that the absence of ATG5 abolishes

the MTX-induced aggregation of GFP-LC3 in cytoplasmic puncta, yet does not affect MTX-

induced nuclear shrinkage. C-G. U2OS cells were left untreated (UNT) or incubated for 18 h with 2

µM MTX or 300 µM oxaliplatin (OXA) and then characterized for GFP-LC3 aggregation and

apoptotic chromatin condensation (by automated image analysis, C,D), ATP release in the

supernatant (by an enzymatic assay, E), surface calreticulin (CRT) exposure (upon

immunofluorescence on intact cells and cytofluorometry, F), and and HMGB1 release in the

supernatant (by ELISA, H). Results are reported as means ± SD of n = 3 independent

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determinations. Asterisks indicate significant (*p<0.05, unpaired Student’s t test) effects of the

knockdown of autophagy-relevant gene products, compared to siUNR-transfected cells treated with

the same chemotherapeutic agent.

Supplemental Figure 5. Effects of autophagy inhibitors and inducers on extracellular ATP

concentrations. A,B. Human osteosarcoma U2OS cells were left untreated (UNT) or treated

overnight with the indicated combinations of mitoxantrone (MTX, 2 µM), oxaliplatin (OXA, 300

µM), hydroxychloroquine (HCQ, 20 µM), bafilomycin A1 (BafA1, 50 nM) and/or carbobenzoxy-

valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk, 100 µM), followed by ATP

quantification in the supernatants with a luciferase-based enzymatic assay. Results are reported as

means ± SD of n = 3 independent determinations. Asterisks indicate significant (p<0.05, Student’s t

test) effects from HCQ, BafA1 and or Z-VAD-fmk, as compared to cells treated with MTX or OXA

alone. C. Representative LC3 distribution in U2OS cells stably expressing a GFP-LC3 fusion

protein that were either left untreated (UNT) or incubated with 1 µM rapamycin (RAPA) for 18 h.

Quantitative data are reported. D. ATP quantification in the supernatant of U2Os cells treated as in

C.

Supplementary Figure 6. Autophagy does not affect the kinetic of cell death induced by

chemotherapy. Murine CT26 (A,B) and MCA205 (C,D) cells were transiently transfected with a

control siRNA (siUNR) or with siRNAs targeting Atg5 (siAtg5), Atg7 (siAtg7) or Beclin 1

(siBcn1) for 48 h, and then cultured in the presence of 1 μM mitoxantrone (MTX) (A,C) or 150 µM

oxaliplatin (OXA) (B,D) for the indicated time. Cell death-related parameters were monitored by

cytofluorometry upon AnnexinV/PI co-staining. Black and white columns report the percentage of

dead (AnnexinV+ PI+) and dying (AnnexinV+ PI-) cells, respectively.

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Supplementary Figure 7. Mitoxantrone-treated, autophagy-deficient cells fail as dead-cell

tumor vaccines. Murine CT26 (A) or MCA205 cells (B) transfected with a control (SCR) or with

Atg5- or Atg7-targeting shRNAs were cultured overnight in the presence of mitoxantrone (MTX, 2

and 1 µM for CT26 and MCA205 cells, respectively) and then subcutaneously injected into the

flank of syngenic BALB/c (A) or C57BL/6 (B) mice. PBS was injected instead of dying cells as a

control conditions. One week later, mice were re-challenged with living cells of the same type and

tumor incidence was monitored (n = 10 mice per group). Kaplan-Meier curves were statistically

evaluated by the logrank test (*p<0.05, as compared to SCR-transfected, MTX-treated cells).

Supplementary Figure 8. UV-treated, autophagy-deficient MCA205 cells fail to vaccinate

C57BL/6 mice. A,B. MCA205 cells were left untransfected (WT) or transfected with a control

siRNA (siUNR) or with siRNAs for the depletion of the indicated proteins for 48 h and then left

untreated (Co), irradiated with UV (1.5 kJ/m2) (A) or incubated with 1 µM mitoxantrone (MTX)

overnight (B). Washed cells (or an equal volume of vehicle, PBS) were subcutaneously injected into

the lower flank of C57BL/6 mice, and one week later living MCA205 cells were inoculated into the

contralateral flank. Tumor incidence was monitored for 60 days in groups of 10 mice for each

treatment. Kaplan-Meier curves in A were statistically evaluated by the logrank test (*p<0.05, as

compared to siUNR-transfected UV-treated or MTX-treated cells, as appropriate). Panel B reports

tumor incidence in C57BL/6 mice at the end of the experiment (60 days after the challenge with

living cells) C. MCA205 cells were cultured overnight with 1 µM MTX alone or in the presence of

20 µM hydroxychloroquine (HCQ) or 50 nM bafilomycin A1 (BafA1). Then, cells were evaluated

for their capacity to vaccinate against cancer cell growth as in A. Kaplan-Meier curves were

statistically evaluated by the logrank test (*p<0.05, as compared to UV- or MTX-treated cells, as

appropriate).

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Supplementary Figure 9. Recombinant calreticulin fails to restore defective immunogenicity

in autophagy-deficient cells treated by chemotherapy. A. CT26 cells were incubated with 2 μM

mitoxantrone (MTX) or with an equivalent volume of PBS overnight, and then coated with

recombinant calreticulin (rCRT, 3 μg per 106 cells) for 20 min at 4°C. After extensive washing, the

percentage of cells exhibiting surface CRT was quantified by indirect immunofluorescence and

cytofluorometry upon the exclusion of dead cells from the analysis. B,C. Murine CT26 (B) or

MCA205 cells (C) were left untransfected (WT) or transiently transfected with a control siRNA

(siUNR) or with siRNAs targeting Beclin 1 (siBcn1) for 48 h, and then maintained in the absence or

in the presence of 2 or 1 μM mitoxantrone (MTX) overnight, respectively. Cells were then coated or

not with rCRT as in A, washed and subcutaneously injected into the lower flank of BALB/c (B) or

C57BL/6 mice (C). Tumor incidence was monitored for 60 days. Kaplan-Meier curves were

statistically evaluated by the logrank test (*p<0.05, as compared to MTX-treated cells). D.

Percentage of BALB/c mice that remained tumor-free 60 days after the inoculation of living cells.

The graph summarizes results from B, as well as an additional control. The knockdown of CRT

with a specific siRNA reduces the vaccination potential of MTX-treated CT26 cells, and this effect

is reversed by coating them with rCRT (*p<0.05, χ2 test).

Supplementary Figure 10. Autophagy-deficient tumors fail to respond to immunogenic

chemotherapy in vivo. SCR-transfected (SCR), Atg5-deficient (Atg5KD) and Atg7-deficient

(Atg7KD) murine fibrosarcoma MCA205 cells were inoculated into the flank of immunodeficient

(nu/nu) (A,C) or immunocompetent C57BL/6 mice (B). When tumor area reached 40-80 mm2, mice

were assigned to homogenous groups (5 mice each), which were treated with either 1.7 mg/kg

intratumoral mitoxantrone (MTX) (A) or 5 mg/kg intraperitoneal MTX (B,C). An equivalent

volume of PBS was administered as negative control (A-C). Tumor surface was assessed every 2-3

days. Representative experiments are shown, each of which has been repeated at least twice,

yielding similar results. Results are means ± SEM (*p<0.05, unpaired Student’s t test). 

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Supplementary Figure 11. Autophagy incompetence of Atg5-deficient (Atg5KD) and Atg7-

deficient (Atg7KD) in vivo. SCR-transfected (SCR, control cells), Atg5KD and Atg7KD murine CT26

cells were inoculated into the flank of BALB/c mice and tumors were allowed to develop. When the

tumor area reached 40-80 mm2, mice were treated with 5 mg/kg intraperitoneal mitoxantrone

(MTX) or an equivalent volume or PBS (vehicle control) followed by the immunohistochemical

detection of LC3 aggregation. Representative microphotographs are shown.

Supplementary Figure 12. Autophagy-deficient tumors undergo apoptosis and necrosis in

response to systemic chemotherapy in vivo. SCR-transfected (SCR, control cells), Atg5-deficient

(Atg5KD) and Atg7-deficient (Atg7KD) murine fibrosarcoma MCA205 cells were inoculated into the

flank of syngenic C57BL/6 mice. When tumor area reached 25 mm2, mice received one

intraperitoneal injection of mitoxantrone (MTX, 5 mg/kg) or vehicle (PBS) (time 0), and tumors

were recovered 24, 48 or 72 h after treatment, followed by fixation, nuclear counterstaining with 1

μM Hoechst 33342 (A,B) and indirect immunofluorescence detection of HMGB1, when required by

the experimental layout (C,D). Representative images of cells exhibiting MTX-induced chromatin

condensation and HMGB1 release from non-condensed nuclei are shown in panels A and C,

respectively. Quantitative data are reported in B and D, respectively, (means ± SEM, n = 10 tissue

sections from distinct tumors). Asterisks indicate significant (*p<0.05, unpaired Student’s t test)

induction of apoptosis or necrosis by MTX as compared to PBS control.

Supplementary Figure 13. The ecto-ATPase inhibitor ARL67156 stimulates the recruitment

of immune system effectors. SCR-transfected (SCR, control cells) or Atg5-deficient (Atg5KD)

murine colon carcinoma CT26 cells were inoculated in the flank of BALB/c mice and allowed to

generate palpable tumors. At that stage, mice were treated with 5 mg/kg intraperitoneal

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mitoxantrone (MTX), in combination with 1.6 mg/kg intratumoral ARL67156 (ARL) in 100 µL

PBS (or an equivalent volume of PBS). Nine days after treatment, tumors were recovered, single

cell suspensions were obtained and cells were stained with antibodies specific for the indicated

proteins and analyzed by cytofluorometry. Results are reported as means ± SEM, n = 10 distinct

tumors (*p<0.05, **p<0.01, ***p<0.001, unpaired Student’s t test).

Supplementary Figure 14. Ecto-ATPase inhibitors restore the efficiency of immunogenic

chemotherapy in autophagy-deficient tumors. SCR-transfected (SCR, control cells), Atg5-

deficient (Atg5KD) and Atg7-deficient (Atg7KD) MCA205 cells were inoculated into the flank of

immunocompetent C57BL/6 (A,C) or isogenic, immunodeficient (nu/nu) mice (B,D). When tumor

area reached 40-80 mm2 (day = 0), mice were assigned to homogenous groups (5 mice each), which

were treated with 5 mg/kg intraperitoneal mitoxantrone (MTX) alone or in combination with 1.6

mg/kg intratumoral ARL67156 (ARL) at day 0 and 3. PBS was injected intraperitoneally or

intratumorally as a vehicle control instead of MTX or ARL67156, respectively. Tumor surface was

assessed every 2-3 days. Representative experiments are shown in A and B (means ± SEM, n = 5

mice), each of which has been repeated at least twice, yielding similar results. Results from pooled

experiments are shown in C and D. Statistical significance was estimated with unpaired Student’s t

test (*p<0.05, as compared to ARL-PBS treated mice). Alternatively, CT26 cells were inoculated

into the flank of immunocompetent BALB/c (E) or isogenic, immunodeficient (nu/nu) mice (F).

When tumor area reached 40-80 mm2 (day = 0), mice were treated with 5 mg/kg intraperitoneal

MTX alone or in combination with 1.6 mg/kg intratumoral ARL67156 (ARL) or 12.5 mg/kg

NGXT191 (NGXT) at day 0 and 3. PBS was employed as a control as indicated above. Data are

reported as means ± SEM of 5 mice (*p<0.05, **p<0.01, unpaired Student’s t test, as compared to

PBS control).

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Supplementary Figure 15. Ecto-ATPase inhibitors restore the efficiency of immunogenic,

oxaliplatin-based chemotherapy in autophagy-deficient tumors. Murine CT26 (A,C) or

MCA205 cells (B) stably transfected with a control shRNA (SCR) or with a shRNA for the

depletion of Atg5 (Atg5KD) were inoculated into immunocompetent BALB/c (A),

immunocompetent C57BL/6 (B) or immunodeficient (nu/nu) mice (C) and allowed to generate

tumors. When tumor area reached 40-80 mm2 (day = 0), mice were treated with 10 mg/kg

intraperitoneal oxaliplatin (OXA) alone or in combination with 1.6 mg/kg intratumoral ARL67156

(ARL). PBS (vehicle control) was injected intraperitoneally or intratumorally, as appropriate. Data

are reported as means ± SEM, n = 5 animals per group (*p<0.05, **p<0.01, unpaired Student’s t

test).

Supplementary Figure 16. Tumors engineered to express the ecto-ATPase CD39 fail to

respond to immunogenic chemotherapy. A. Immunofluorescence detection of CD39 in MCA205

cells transfected with a CD39-encoding or control (Co) plasmid. Unspecific fluorescence by

secondary antibodies was monitored with an appropriate isotype control (grey) B. Wild-type (WT)

or CD39-expressing MCA205 cells were cultured in the presence of 1 µM mitoxantrone MTX or an

equivalent volume of PBS (control) for 18 h and inoculated into the flank of immunocompetent

C57BL/6 mice, which one week later were re-challenge with live tumor cells of the same type.

Tumor incidence was monitored for the 60 days. C. Control or CD39-expressing MCA205 tumors

growing on immunocompetent C57BL/6 (C) or immunodeficient (nu/nu) mice (D) were treated

with 5 mg/kg intraperitoneal MTX alone or in combination with 1.6 mg/kg intratumoral ARL67156

(ARL) or with vehicle (PBS) alone, as indicated. The treatment started when the tumor area reached

40-80 mm2 (day = 0).Data are reported as means ± SEM (n = 5 animals per group, *p<0.05,

**p<0.01, unpaired Student’s t test).

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Suppl. Fig. 1

Atg5

β-Actin

SCR52

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MW(kDa)

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CRT

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D

Hoechst anti-LC3

Page 31: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

A

Suppl. Fig. 2

Atg5

β-Actin

SCR 52

32

MW(kDa)

56

42 β-Actin

Atg7

7141

SCR

MW(kDa)

75

42

UNT MTX OXA

SC

RA

tg5K

DA

tg7K

D

Hoechst anti-LC3 10 μm

B

0

5

10

15

25

20

SCRUNTMTXOXA

Cou

nts

Atg5KD

0

5

10

15

25

20

CRT100 101 102 103 104

Atg7KD

0

5

10

15

25

20

E

CSCR Atg5KD Atg7KD

10

20

30

0

40

LC3

punc

ta/c

ell

** * *

D

Ext

race

llula

r ATP

[10-1

0 M

]

1

2

0

3

* * * *

F

CR

T+ ce

lls [%

]

5

10

15

20

0

25

G

Cel

ls [

%]

20

40

60

80

0

100AnnV+PI+AnnV+PI-

H

HM

GB

1 [n

g/m

L]

20

40

60

0

80

UN

T

MTX

OX

A

UN

T

MTX

OX

A

UN

T

MTX

OX

A

Page 32: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

Suppl. Fig. 3

UNT MTX OXA

CRT

102030

0

5040

Cou

nts

101 102 103100 104

Atg5-/-

Atg7-/-

WTUNTMTXOXA

102030

0

5040

Cou

nts

102030

0

5040

Cou

nts

A

MTX OXA RAPA

WT Atg5-/- Atg7-/-

LC3-ILC3-II

1614

42

MW(kDa)

β-Actin

UNT MTX OXA RAPAUNT UNT MTX OXA RAPA

MW(kDa)

56

42 β-Actin

Atg7

MW(kDa)

75

42

Atg7-/-WT

Atg5

β-Actin

Atg5-/-WT

B

C D

WT

Atg

7-/-

10 μmanti-LC3Hoechst

Atg

5-/-

Page 33: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

A

Suppl. Fig. 4

siUNR

siATG5

siBCN1

siATG7

ATG5

BCN1

ATG7

56

75

60

*

MW(kDa)

D

E

CsiUNRsiATG5siATG7siATG10siBCN1

5

10

15

0

20

LC3

pun

cta/

cell

UNT MTX OXA

** *

*

* * * *

F

Ext

race

llula

r ATP

[10-

8 M

]

5

10

15

0

20

UNT MTX OXA

**

**

*

*

**

20

40

0

60

CR

T+ c

ells

[%]

UNT MTX OXAG

20

40

0

50

HM

GB

1 [n

g/m

L]

UNT MTX OXA

1

2

3

0

4

Nuc

lear

Are

a [A

.U.]

UNT MTX OXA

siUNRsiATG5siATG7siATG10siBCN1

B UNT

10 μmHoechst GFP-LC3

siATG5

siUNR

10

30

MTX

Page 34: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

Suppl. Fig. 5

C

5

10

15

0

20

GFP

-LC

3 pu

ncta

/cel

l

UN

T

RA

PA10 μm

GFP-LC3Hoechst

UNT

RAPA

Ext

race

llula

r ATP

[10-9

M]

D

0

2

4

6

8

10

UN

T

MTX

RA

PA

*

5

10

0

15

MTX OXA

Co

Baf

A1

HC

Q Co

Baf

A1

HC

Q Co

Baf

A1

HC

Q

Ext

race

llula

r ATP

[10-9

M]

*

A

5

10

0

15

Z-VA

D-fm

k

Co

Z-VA

D-fm

k

Co

MTX OXA

Z-VA

D-fm

k

Co

Ext

race

llula

r ATP

[10-9

M]

**

B

Page 35: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

MTXsiUNR siAtg5 siAtg7 siBcn1 siUNR siAtg5 siAtg7 siBcn1

siUNR siAtg5 siAtg7 siBcn1

A B

C D

OXA

MTX OXA

Time [h]

AnnexinV+PI-

AnnexinV+PI+

Time [h] Time [h]12 24 48 12 24 48 12 24 48 12 24 48

0

20

40

60

80

100

CT2

6 ce

lls [%

]

12 24 48 12 24 48 12 24 48 12 24 48

20

40

60

80

0

100

CT2

6 ce

lls [%

]

12 24 48 12 24 48 12 24 48 12 24 48

20

40

60

80

0

100

MC

A20

5 ce

lls [%

]

siUNR siAtg5 siAtg7 siBcn1

Time [h]12 24 48 12 24 48 12 24 48 12 24 48

0

20

40

60

80

100

MC

A20

5 ce

lls [%

]

Suppl. Fig. 6

Page 36: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

Suppl. Fig. 7

A B

0

20

40

60

80

100

20 400 60Days after rechallenge

SCR + MTX

PBSTum

or-fr

ee B

ALB

/c m

ice

[%]

Atg5KD + MTXAtg7KD + MTX

20

40

60

80

0

100

20 400 60Days after rechallenge

SCR + MTX

PBS

Tum

or-fr

ee C

57B

L/6

mic

e [%

]

Atg5KD + MTXAtg7KD + MTX

* *

Page 37: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

Suppl. Fig. 8

Tum

or-fr

ee C

57B

L/6

mic

e [%

]

0

20

40

60

80

100

MTX

n=50

n=50n=50

n=15n=35

n=35 n=15

n=35**

* **

B

A

WT UV

siUNR+UV

siBcn1+UV

PBS

Days after rechallenge

20

40

60

80

0

100

20 400 60

Tum

or-fr

ee C

57B

L/6

mic

e [%

]*

MTX

MTX+BafA1

MTX+HCQPBS

C

20

40

60

80

0

100

20 400 60

Tum

or-fr

ee C

57B

L/6

mic

e [%

]

Days after rechallenge

*

PB

S Co

siU

NR

siA

tg3

siA

tg5

siA

tg7

siA

tg10

siB

cn1

Page 38: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

20

40

60

80

0

100

Surfa

ce C

RT+ /P

I- cel

ls [%

]

Suppl. Fig. 9

A B

PBS MTX rCRT MTX+rCRT

Tum

or-fr

ee B

ALB

/c m

ice

[%]

WT+MTXsiUNR+MTX

siBcn1+MTXsiBcn1+MTX+rCRTPBS

40

60

80

0

100

20 400 60Days after rechallenge

20

*Tu

mor

-free

BA

LB/c

mic

e [%

]

PBS MTXsiUNR

rCRTCosiCRT siBcn1

rCRTCorCRTCo

*

20

40

60

80

0

100

Tum

or-fr

ee C

57B

L/6

mic

e [%

]

20 400 60Days after rechallenge

20

40

60

80

0

100

WT+MTXsiUNR+MTX

siBcn1+MTXsiBcn1+MTX+rCRT

PBS

*

C D

MTX

n=10

n=10n=10 n=10

n=22

n=13

n=10n=10

**

*

Page 39: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

Suppl. Fig. 10

A

Days after treatment

Tum

or a

rea

[102

mm

2 ]

4

32

1

0 5 10 15 20 25

5

*

SCR

4

32

1

0 5 10 15 20 25

5

*

Atg5KD

4

32

1

0 5 10 15 20 25

5

*

Atg7KD

PBSMTX IT

B

Days after treatment

Tum

or a

rea

[102

mm

2 ]

4

32

1

5

5 10 15 20 25

*

SCR

4

32

1

5

0 5 10 15 20 25

Atg5KD

4

3

2

1

0 5 10 15 200 25

Atg7KD

PBSMTX

C

Days after treatment

Tum

or a

rea

[102

mm

2 ] 4

3

2

1

0 5 10 15 20 25

SCR4

3

2

1

0 5 10 15 20 25

Atg5KD

4

3

2

1

0 5 10 15 20 25

Atg7KD

PBSMTX

Page 40: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

SC

RA

tg5K

DA

tg7K

D

MTXPBS

Suppl. Fig. 11

20 μm

10 μm

Page 41: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

Suppl. Fig. 12

SCR PBS SCR MTX

Atg5KD MTX Atg7KD MTX

10 μmHMGB1-GFPHoechst Time [h]

HM

GB

1- nuc

lei [

%]

48 72 24 48 72 24 4824 720

20

40

60

80

100PBSMTX

SCR Atg5KD Atg7KD

SCR PBS SCR MTX

Atg5KD MTX Atg7KD MTX

10 μm

Hoechst 33342 24 48 72 24 48 72 24 48 72

10

20

30

40

0

50

PBSMTX

Apo

ptot

ic n

ucle

i [%

]SCR Atg5KD Atg7KD

Time [h]

A B

C D

*

**

**

**

**

*

*

**

**

*

**

10 μmanti-HMGB1Hoechst 33342

Page 42: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

Fig. Suppl. 13

ns*** *

050

100150200250

Tum

or a

era

[mm

2 ]

SCR

PB

S

AR

L

PB

S

AR

L

Atg5KD

0

5

10

15

CD

4+ cel

ls [%

]

02468

10

CD

8+ cel

ls [%

]

0.5

1.0

1.5

0.0

2.0

TCR

+ cel

ls [%

]

0.5

1.0

0.0

1.5

+ IL

17A+ c

ells

[%]

02468

10

CD

8+IF

N+

cel

ls [%

]

2

4

0

6C

D4+

IFN+

cel

ls [%

]

SCR

PB

S

AR

L

PB

S

AR

L

Atg5KD

ns*** ***

ns* *

ns* ***

ns* *

ns* *

ns** *

Page 43: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

A B

Tum

or a

rea

[102

mm

2 ] 4

3

2

1

0 5 10 15 20 25

4

3

2

1

0 5 10 15 20 25

4

3

2

1

0 5 10 15 20 25Days after treatment

Tum

or a

rea

[102

mm

2 ]

4

3

2

1

0 5 10 15 20 25

4

3

2

1

0 5 10 15 20 25

3

2

1

0 5 10 15 20 25

5

Days after treatment

C

PBSMTX

0123456

ARLAtg5KD Atg7KDSCR

C57BL/6 WT

* **

*

- + - + - +

Tum

or a

rea

[102

mm

2 ]

D

* **

Suppl. Fig. 14

PBS + ARLMTX + ARL

PBS + ARLMTX + ARL

0123456 PBS

MTX

ARL

C57BL/6 nu/nu

- + - + - +

Tum

or a

rea

[102

mm

2 ]

Atg5KD Atg7KDSCR

Atg5KD Atg7KDSCR Atg5KD Atg7KDSCR

E

01234567

Atg5KD Atg7KDCT26 SCR

BALB/c WT

ARL NGXT

- + - - + - - + -- - + - - + - - +

Tum

or a

rea

[102

mm

2 ]

* * ** *

***

PBSMTX

F

01234567

Atg5KD Atg7KDCT26 SCR

BALB/c nu/nu

ARL NGXT

- + - - + - - + -- - + - - + - - +

Tum

or a

rea

[102

mm

2 ] PBSMTX

Page 44: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

1

2

3

0

4Tu

mor

are

a [1

02 m

m2 ]

Days after treatment

SCR Atg5KD

5 10 15 200 25Days after treatment

*

5 10 15 200 25

**

PBSPBS+OXA ARL+OXA

1

2

3

0

4

0 5 10 15 20 25Days after treatment

0 5 10 15 20 25Days after treatment

***

**

PBSPBS+OXA ARL+OXA

Tum

or a

rea

[102

mm

2 ]

SCR Atg5KD

1

2

3

4

0

5

0 5 10 15Days after treatment

0 5 10 15Days after treatment

PBSPBS+OXA ARL+OXA

Tum

or a

rea

[102

mm

2 ]

SCR Atg5KD

A

B

C

Fig. Suppl. 15

Page 45: Supporting Online Material for - Science4 by incubating the membranes for 1 h in 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk or bovine serum albumin, followed

CD39+101 102 103100 104 101 102 103100 104

0

30

60

90

150

130

Co CD39

Cou

nts

2.05 % 92.86 %

Co CD39

Tum

or a

rea

[102

mm

2 ]

0

1

2

3

4

5 10 15 200 25Days after treatment

*

0 5 10 15 20 25Days after treatment

**

A B

C

D

Fig. Suppl. 16

5 10 15 200Days after treatment

Tum

or a

rea

[102 m

m2 ]

0

1

2

3

4

5 10 15 200Days after treatment

5 10 150 20Days after treatment

5 10 150 20Days after treatment

Tum

or-fr

ee C

57B

L/6

mic

e [%

]20 400 60

Days after rechallenge

WT+MTX

WT+PBS

20

40

60

80

0

100

*

20 400 60Days after rechallenge

CD39+PBS

CD39+MTX

5 10 15 200 25Days after treatment

0 5 10 15 20 25Days after treatment

*

PBSMTX

ARL+PBSARL+MTX

PBSMTX

ARL+PBSARL+MTX

PBSMTX

ARL+PBSARL+MTX

PBSMTX

ARL+PBSARL+MTX

Co CD39

IsotypeantiCD39