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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)
1
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
4
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
5
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).
6
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
7
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.
8
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
9
(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
10
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
11
(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-
12
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).
13
Supplementary References
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a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3, 452-460 (2007).
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autophagy. Proc Natl Acad Sci U S A 105, 3374-3379 (2008).
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1
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
2
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
3
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.
4
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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.
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
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.
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).
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).
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
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).
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).
Suppl. Fig. 1
Atg5
β-Actin
SCR52
17
MW(kDa)
56
42 β-Actin
Atg7
717
745
SCR
MW(kDa)
75
42
A
B
5
10
15
0
25
20
Cou
nts
101 102 103100 104
Atg5KD
Atg7KD
SCRUNTMTXOXA
5
10
15
0
25
20
Cou
nts
5
10
15
0
25
20
Cou
nts
UNT MTX OXA
10 μm
C
CRT
SC
RA
tg5K
DA
tg7K
D
Hoechst anti-LC3
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
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-/-
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
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
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
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
* *
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
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
**
*
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
SC
RA
tg5K
DA
tg7K
D
MTXPBS
Suppl. Fig. 11
20 μm
10 μm
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
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** *
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
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
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