www.sciencemag.org/cgi/content/full/337/6102/1678/DC1

Supplementary Materials for

An Immunosurveillance Mechanism Controls Cancer Cell Ploidy

Laura Senovilla, Ilio Vitale, Isabelle Martins, Maximilien Tailler, Claire Pailleret, Mickaël Michaud, Lorenzo Galluzzi, Sandy Adjemian, Oliver Kepp, Mireia Niso-

Santano, Shensi Shen, Guillermo Mariño, Alfredo Criollo, Alice Boilève, Bastien Job, Sylvain Ladoire, François Ghiringhelli, Antonella Sistigu, Takahiro Yamazaki, Santiago

Rello-Varona, Clara Locher, Vichnou Poirier-Colame, Monique Talbot, Alexander Valent, Francesco Berardinelli, Antonio Antoccia, Fabiola Ciccosanti, Gian Maria Fimia,

Mauro Piacentini, Antonio Fueyo, Nicole L. Messina, Ming Li, Christopher J. Chan, Verena Sigl, Guillaume Pourcher, Christoph Ruckenstuhl, Didac Carmona-Gutierrez,

Vladimir Lazar, Josef M. Penninger, Frank Madeo, Carlos López-Otín, Mark J. Smyth, Laurence Zitvogel,* Maria Castedo,* Guido Kroemer*

*To whom correspondence should be addressed. E-mail: kroemer@orange.fr (G.K.); castedo@igr.fr (M.C.); zitvogel@igr.fr (L.Z.)

Published 28 September 2012, Science 337, 1678 (2012)

DOI: 10.1126/science.1224922

This PDF file includes:

Materials and Methods Figs. S1 to S15 Tables S1 to S3 References

1

Materials and Methods

Unless otherwise indicated, media 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).

Antibodies. Rabbit polyclonal antibodies against CRT (ab2907) and ERp57 (ab10287) and a rat

monoclonal antibody against CD112 (Nectin 2; ab16912) were purchased from Abcam,

(Cambridge, UK). Conjugated antibodies against CD47 (FITC anti-mouse CD47, 127503), MHC

class I (FITC anti-mouse H2Kd, 116605), CD155 (PE anti-mouse CD155 (PVR), 131508), the

corresponding isotype controls (PE mouse IgG2a, 400507; FITC mouse IgG2a, 400505) as well

as a purified anti-mouse CD120a (TNFRI/p55) antibody (113001) were obtained from

BioLegend (San Diego, USA). Purified anti-mouse CD95 (Fas, 554254), and FITC- and PE-

conjugated antibodies specific for CD8a and CD4, respectively, were from BD Pharmingen (San

Jose, USA). Rabbit polyclonal antibodies against PERK (mAB 3192), eIF2� (9722),

phosphorylated PERK (Thr980) (mAB, 3179), anti-phospho-eIF2������������ ), and GADD45

(3518) were from Cell Signaling Technology (Danvers, USA). Purified anti-mouse CD262

(DR5/TRAIL R2, 14-5883) and FITC-conjugated secondary antibodies anti-Armenian hamster

IgG (11-4111) were from eBioscience (San Diego, USA). Pan specific-antimouse Rae-1

(MAB17582), anti-human MICA/B (MAB13001) and anti-human ULBP1 (MAB1380)

antibodies were from R&D Systems (Minneapolis, USA). The mouse monoclonal antibody

against human HLA class I (H1650) was purchased from Sigma-Aldrich. The anti-�������-actin

antibody (MAB1501) was from Millipore (Temecula, CA, USA). Antibodies for the in vivo

depletion of CD4+ and CD8+ cells, GK1.5 (BE0003-1) and 2.43 (BE0061), respectively, were

purchased from Bioxcell (New Hampshire, US).

2

Cell lines and culture conditions. All cell lines were cultured at 37ºC under 5% of CO2, in the

appropriate medium containing 10% fetal bovine serum (FBS) and 100 U mL-1 penicillin sodium

and 100 µg mL-1 streptomycin sulfate. Cell type-specific culture conditions include: Dulbecco’s

modified Eagle’s medium (DMEM) supplemented as above plus 1 mM sodium pyruvate for

murine Lewis lung carcinoma (LLC) cells; DMEM supplemented as above plus 1 mM sodium

pyruvate and 10 mM HEPES buffer for human osteosarcoma U2OS cells; DMEM supplemented

as above plus 1 mM sodium pyruvate, 10 mM HEPES buffer and 1% non-essential amino acids

for wild type (WT), Bax-/-Bak-/-, eIF2�S51A and Casp8-/- mouse embryonic fibroblasts (MEFs);

DMEM/F12 (1:1) medium supplemented as above plus 1 mM sodium pyruvate and 10 mM

HEPES buffer for human non-small cell lung carcinoma A549 cells; DMEM/M199 (4:1) medium

supplemented as above plus 0.01 mg mL-1 hygromycin B for hTERT-immortalized human

foreskin BJ5ta cells; RPMI 1640 medium supplemented as above for human colon carcinoma

DLD-1 cells; RPMI 1640 medium supplemented as above plus 200 �g mL-1 G418 for trisomic

human colon carcinoma DLD-1+7 cells (25); RPMI 1640 medium supplemented as above plus 1

mM sodium pyruvate and 1 mM HEPES buffer for murine colon carcinoma CT26 and murine

fibrosarcoma MCA205 cells; McCoy’s 5A modified medium (PAA Laboratories GmbH,

Pasching, Austria) supplemented as above plus 1 mM sodium pyruvate and 10 mM HEPES for

human colon carcinoma HCT 116 cells.

For clonogenicity experiments, cells were treated for 48 h with 100 nM nocodazole followed by

the cloning of cells characterized by an 8n DNA content on a FACSVantage cell sorter (BD

Biosciences San José, USA). Clonogenicity was evaluated 2 weeks after sorting.

Generation of hyperploid clones. Parental diploid cells were treated for 48 h with 0.6 µg mL-1

cytochalasin D, 2 µM dihydrocytochalasin B (DCB) (Calbiochem, San Diego, USA) or 100 nM

3

nocodazole and then cultured for 2 weeks in drug-free culture medium, followed by cloning of

cells characterized by an 8n DNA content on a FACSVantage cell sorter (BD Biosciences), as

previously described (35).

Isolation and culture of primary cells. Intestinal epithelial cells (IECs). The isolation of IECs

was performed from 8 – 9 weeks old male Tp53-/- C57Bl/6 mice. Quickly after sacrifice, colons

were surgically removed, longitudinally opened and washed in 100 mM Ca2+- and Mg2+-free

PBS. Colons were then washed in Ca2+- and Mg2+-free Hank’s Balance Salt Solution (HBSS)

supplemented with 2% glucose, 25 ng mL-1 amphotericine B, 100 U mL-1 penicillin sodium and

100 µg mL-1 streptomycin sulfate, and cut into 1-mm pieces. Thereafter, they were dissociated by

incubation for 10 min at room temperature (with occasional vigorous shaking) in Ca2+- and Mg2+-

free HBSS supplemented with 0.2 mg mL-1 soybean trypsin inhibitor (SBTI), 2% bovine serum

albumin (BSA), 20 µg mL-1 dispase I and 50 µg mL-1 collagenase XI, after which DMEM

supplemented with 10% sorbitol, 100 U mL-1 penicillin sodium, 100 µg mL-1 streptomycin

sulfate and 5% FBS was added. Cells and tissue debris were vigorously shaken and sediments

were removed. Cells were recovered by centrifugation (3 min at 120 g) and washed 4 times with

DMEM + 2% sorbitol. Then, IECs were seeded in 25 cm2 flasks pre-coated with rat tail collagen,

type I (BD Bioscience), and cultured in phenol red-free DMEM/F12 medium supplemented with

5 µg mL-1 insulin, 0.2% glucose, 50 nM dexamethasone, 2% FBS, 6 nM sodium selenite, 50 nM

triiodo-thyronine sodium, 10 µg mL-1 epithelial growth factor (EGF), 5 µg mL-1 transferrin, 20

µM HEPES, 2 mM L-glutamine and 100 U mL-1 penicillin sodium and 100 µg mL-1 streptomycin

sulfate.

Mammary gland cells (MGCs). The isolation of primary mammary gland epithelial cells was

performed on 8–9 week old female Tp53-/- C57Bl/6 mice, as previously described (36). After

4

sacrifice, inguinal mammary glands were surgically removed and washed in DMEM/F12 medium

supplemented with 100 U mL-1 penicillin sodium������� mL-1 streptomycin sulfate ���������

mL-1 gentamycin. Thereafter, they were dissociated by incubation for 30 min at 37°C (with

occasional vigorous shaking) in wash medium further supplemented with 0.15% type A

collagenase (Roche Diagnostics GmbH, Mannheim, Germany). Dissociated cells were cultured in

flasks that had been pre-coated with 0.1% gelatin (4 h, 37°C). MGCs were cultured in

����������� �����!!"����#���$ #%��&��'�������g mL-1 ���#��() ����"*�#�������g mL-1

insulin and 5 ng mL-1 epithelial growth factor (EGF).

Colonic crypts. The isolation of primary colonic crypts was performed on 8-9 week old female

Tp53-/- C57Bl/6 mice. Crypts were isolated and maintained in culture as described by Sato et al.

(37, 38).

Splenic lymphocyte isolation. After euthanasia, spleens were rapidly removed and placed into

RPMI medium supplemented with 100 U mL-1 penicillin sodium and ����� mL-1 streptomycin

sulfate. Splenic lymphocytes were obtained upon mechanical pressure to the organ, elimination

of debris and elimination of erythrocytes by means of the RBC lysis buffer (42301, BioLegend).

After isolation, cells were incubated with FITC-conjugated anti-CD8a and PE-conjugated anti-

CD4 antibodies (5µg mL-1 in 200 µL of PBS supplemented with 2% BSA (w/v)) for 30 min at

4°C, washed and analyzed by cytofluorometry.

Plasmid construction and siRNAs. For the construction of a plasmid encoding a CRT variant

constitutively exposed on the plasma membrane (mCRT), the cDNA corresponding to the mouse

CRT sequence (NM_007591) was obtained by RT-PCR on an RNA extract from CT26 cells with

the primers 5’-TATACCCGGGCTCCTTTCGGTGCCGCTC and 5’-

5

TATACCGCGGGGCTTGGCCAGGGGATTC. This sequence was cloned into the pDisplay

vector (Invitrogen) using SmaI and SacII restriction sites. With the aim to avoid an immunogenic

effect in in vivo experiments, the hemaglutinin A epitope as well as the KDEL C-terminal

sequence of CRT were deleted. To this aim, a SmaI restriction site was created at the beginning

of the epitope sequence by directed mutagenesis, using the QuickChange® Lightning Site-

directed Mutagenesis Kit (Agilent Technologies, Santa Clara, USA) with the primers 5'-

CAGGTTCCACTGGTGACTATCCCGGGGATGTTCCAGATTATGCTGGGG-3' and 5'-

CCCCAGCATAATCTGGAACATCCCCGGGATAGTCACCAGTGGAACCTG-3'. SmaI

digestion followed by re-ligation of the plasmid was then employed to remove the hemagglutinin

A epitope-coding sequence. GFP-ATG6- end XBP1-DBD-Venus-encoding plasmids were kindly

provided by Dr. Ron Prywes (Columbia University, New York, USA) and Dr. Junying Yuan

(Massachusetts Institute of Technology, Boston, USA), respectively. siRNA heteroduplexes

specific for CRT (sense strand: 5’-CCGCUGGGUCGAAUCCAAAdTdT-3’), Aurora kinase B

(AURKB) (sense strand 5’-GGUGAUGGAGAAUAGCAGUdTdT-3’), Polo-like kinase 1

(PLK1) (sense strand 5’-CGAGCUGCUUAAUGACGAGdTdT-3’) and MAD2 (sense strand 5’-

ACCUUUACUCGAGUGCAGATT3’) were purchased from Sigma-Proligo (The Woodlands,

US). As a control, a non-targeting siRNA with a sequence unrelated to both human and mouse

genomes was used (UNR, sense strand 5’-GCCGGUAUGCCGGUUAAGUdTdT-3’).

Treatments and transfections. Cells were seeded in 6-, 12-well plates, 25, 75 or 175 cm2 flasks

and allowed to adapt for at least 24 h before experiments. Cells were challenged with the

following pharmacological agents: 2 µM vincristine; 4 µM vinblastine; 4 µM colchicine; 6 µM

cytochalasine H; 100 nM nocodazole; 0.6 µg mL-1 cytochalasine D and 2 µM

dihydrocytochalasine B; 100 nM taxotere and 300 nM paclitaxel (Moravek Biochemicals, Inc

6

(Brea, USA). Plasmid transfections were carried out using Lipofectamine 2000® (Invitrogen), as

recommended by the manufacturer. U2OS cells stably co-expressing CRT-GFP and H2B-RFP

were generated by transfection with a CRT-GFP-encoding cDNA and, after selection with G418,

transduction with lentiviral particles expressing H2B-RFP, as previously described (24). U2OS

cells expressing HaloTag® (HT)-CRT were obtained by transfection with a plasmid encoding the

HT-CRT fusion protein (24) and selected with zeocin (Invitrogen). U2OS cell clones stably

expressing GFP-ATF6 or XBP1-DBD-Venus were obtained by plasmid transfection, selection in

800 µg mL-1 G418 (for 2 weeks), and FACS-assisted cloning. mCRT-expressing CT26 clones

were obtained by plasmid transfection and selected with 200 µg mL-1 G418. Transgene

expression in each clone was confirmed by cytofluorometry. For the stable downregulation of

PERK, caspase-8 and ERp57, CT26 cells were infected with retroviral particles carrying the

corresponding shRNA plasmids (20, 39), and several clones were isolated following selection in

0.1 mg mL-1 G418 for 10 days. A scrambled sequence was used as a negative control (shCo). For

the stable downregulation of CRT, CT26 cells were infected with retroviruses carrying the

shCRT plasmid, previously generated using shCRT (OriGene, Rockville, USA) and Phoenix cells

(Orbigen Inc., San Diego, US), and selected with puromycin (10 µg mL-1). CRT downregulation

in each clone was confirmed by immunoblotting. The siRNA-mediated knockdown of CRT,

AURKB, MAD2 and PLK1 was carried out by transfecting cells (at 30-40% confluence) with the

HiPerFect transfection reagent (Qiagen, Hilden, Germany) - previously complexed with 20 nM

siRNA - for 6 to 15 h. siRNA-transfected cells were used for experiments no earlier than 48 h

after transfection.

Automated high-content microscopy and video microscopy. WT human osteosarcoma U2OS

cells or U2OS cells stably transduced with CRT-GFP/H2B-RFP, GFP-ATF6 or XBP1-DBD-

7

Venus (24, 40) were seeded in 96-well imaging plates (BD Falcon), let adapt for 12-24 h and then

treated either with agents from the Institute of Chemistry and Cell Biology (ICCB, Harvard,

USA) known bioactive library (CRT-GFP/H2B-RFP-expressing cells, concentration range: 1-12

µM, from EnzoLifeSciences) or with microtubular and endoplasmic reticulum (ER) toxins (WT,

GFP-ATF6- and XBP1-DBD-Venus-expressing cells). Four-fifteen hours after treatment, cells

were fixed with 4% paraformaldehyde (PFA, w/v in PBS), and either stained wi#%� �� ���

Hoechst 33342 or processed for the immunofluorescence-based detections of phosphorylated

�+���, In both cases, images were acquired using a BD pathway 855 automated microscope (BD

Bioscience). Duplicate acquisitions of approximately 250 cells for each treatment were analyzed

for cellular numbers and average CRT granularity (CRT-GFP/H2B-RFP-expressing cells),

phospho-�+���� �#� � ��� �-.� )�""��� /.�0� !�� ��)"���� #����"�)�# ��� �1�2-ATF6-expressing

cells) or XBP1 upregulation (XBP1-DBD-Venus-expressing cells) (24, 40). For

videomicroscopy, cells were subjected to pulsed observations (every 10 min for up to 48 h) with

the same imaging system. Images were analyzed with the Attovision software (BD Bioscience).

Cytofluorometry. Ecto-CRT was detected by immunofluorescence staining or upon the

expression of a HaloTag-CRT fusion protein, which can be revealed by a means of a cell-

impermeant fluorescent HaloTag ligand, as previously described (20, 24). In some experiments,

Hoechst 33342 (2 µM; Molecular Probes-Invitrogen) was added 1 h earlier to monitor DNA

content. For the assessment of cell cycle distribution, harvested cells were fixed in ice-cold 80%

(v/v) ethanol and stained with 50 �g mL-1 propidium iodide (PI) in 0.1% D-glucose (w/v in PBS)

supplemented with 1 �g mL-1 RNAse A. Primary epithelial cells were labeled with a monoclonal

anti-pan-cytokeratin antibody (C2562, Sigma) - using the same protocol employed for ecto-CRT

detection - prior to classical PI staining. Samples were then analyzed by means of a FACSCalibur

8

or a FACScan cytofluorometer (BD Biosciences). Statistical analyses were carried out by using

the CellQuest™ software (BD Biosciences), upon gating on the events characterized by normal

forward scatter and side scatter values. Epithelial cells were identified by the expression of

cytokeratin.

Immunofluorescence microscopy. For cell surface staining, cells were cultured on 13-mm glass

coverslips in 12-well plates. After treatment, cells were placed on ice, washed twice with PBS

and fixed with 0.25% PFA (w/v in PBS) for 5 min. Cells were then washed twice in PBS and

incubated with primary antibodies (diluted in cold blocking buffer, namely, 5% BSA, v/v in PBS)

for 30 min. Cells were further washed 3 times in ice-cold PBS and then incubated for 30 min with

appropriate secondary antibodies (diluted 1:500 in cold blocking buffer). For intracellular

staining, cells were washed with PBS, fixed with 4% PFA for 20 min, permeabilized with 0.1%

Triton X-100 for 10 min and rinsed three times with PBS. Nonspecific binding sites were blocked

with blocking buffer for 30 min followed by incubation with primary antibodies for 1 h.

Subsequently, cells were washed three times with PBS and incubated for 30 min in FITC or

AlexaFluor® 568-conjugated secondary antibodies (1:1000 in cold blocking buffer; Molecular

Probes-Invitrogen Eugene, USA). When appropriate, 10 µM Hoechst 33342 was used for nuclear

counterstaining. Coverslips were washed with PBS and mounted on slides with 4',6-diamidino-2-

phenylindole (DAPI)-containing Vectashield® mounting medium (Vector Labs, Burlingame,

USA). Fluorescence microscopic assessments were performed with a DMIR2 inverted

fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

Immunoblotting. For immunoblotting, cells were washed with cold PBS at 4ºC and lysed

following standard procedures. Forty µg of proteins were separated according to molecular

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

9

Immobilon polyvinyldifluoride (PVDF) membranes (Millipore, Bedford, USA). Non-specific

binding sites were blocked by incubating membranes for 1 h in 0.05% Tween 20 (v/v in TBS)

supplemented with 5% non-fat powdered milk or BSA. After overnight incubation at 4 ºC,

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

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

SuperSignal West Pico chemoluminescent substrate (Thermo Fisher Scientific, Rockford, USA)

or Amersham ECL+ (GE Healthcare, Little Chalfont, UK). The abundance of � actin was

monitored to ensure equal lane loading.

Isolation of cell surface proteins. Biotinylation and purification of cell surface proteins were

performed using the cell surface protein isolation kit from Pierce-Thermo Scientific (Rockford,

USA), following the manufacturer’s instructions with slight modifications. Briefly, parental and

hyperploid CT26 clones grown on 75 cm2 until 90-95% confluence were placed on ice and

washed twice with ice-cold PBS supplemented with 100 nM Ca2+ and 100 nM Mg2+. Membrane

proteins were then biotinylated by incubating cells for 30 min at 4ºC with Sulfo-NHS-SS-Biotin,

under gentle agitation. The reaction was quenched with the provided quenching solution. Cells

were then gently scraped, transferred to conical tubes and washed twice with TBS. Cell pellets

were lysed in 500 µL lysis buffer containing protease inhibitors (Complete Mini EDTA-free,

Roche) (30 min in ice, vortexing 5 sec every 5 min). Lysates were clarified by centrifugation at

10,000 g for 2 min at 4ºC. Surface proteins were isolated by incubating lysates for 1 h at room

temperature by means of a neutravidin agarose column. Finally, the column was washed three

times with washing buffer supplemented with protease inhibitors, and the surface proteins were

eluted by adding washing buffer containing 1 mM dithiothreitol (DTT).

10

Two-dimensional gel electrophoresis and protein identification by mass spectrometry. After

purification, protein samples from parental and hyperploid cells were precipitated in 5 volumes of

ethanol overnight at 4°C, collected by centrifugation (5000 g, 40 min), and resuspended in

labeling buffer (7 M urea, 2 M thiourea, 2% CHAPS, 1% sulfobetaine SB3-10, 1%

amidosulfobetaine ASB14). Samples were labeled with either the Cy3 or Cy5 dye, while internal

standard samples were labeled with the 3(���(�,�.$��#(�����*�!��#� ��� *��� ��)%� ���!"��$����

labeled with 400 pmol of cyanine dyes in 1 �4� �*� ��%(������ N,N-dimethylformamide (GE

Healthcare) for 30 min on ice under protection from light. The reaction was then quenched with

10 mM lysine for 10 min. A dye-swapping protocol was used to minimize labeling-dependent

artifacts. Quenched Cy3 and Cy5-labeled samples were combined and mixed with an aliquot of

Cy2-labeled standard and an equal volume of 2X sample buffer (7 M urea, 2 M thiourea, 2%

CHAPS, 1% sulfobetaine SB3-10, 1% amidosulfobetaine ASB14, 2% w/v DTT, 2% v/v IPG

buffer 3-10 NL). Isoelectric focusing (IEF) was performed using 18-cm immobilized nonlinear

pH gradient strips (NL IPG pH 3.0 to 10.0; GE Healthcare) on an IPGphor II electrophoresis unit

(GE Healthcare). Proteins were loaded by in-gel rehydration for 9 h using low voltage (30V),

then run according to the following program: 100 V for 1 h, 200 V for 1 h, a progressive increase

up to 1000 V distributed over 30 min, 1000 V for 1 h, a progressive increase up to 8000 V

distributed over 2 h, 8000 V for 4 h. IPG gel strips were reduced in 1% DTT for 10 min at room

temperature and free SH groups were blocked using 4% iodoacetamide (both solutions were

prepared in 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 2% bromophenol blue).

Strips were transferred to 1-mm thick 12% polyacrylamide gels (20 x 20 cm), and the second-

dimension gels were run at 50 mA for 6 h. Electrophoresis procedures were performed in the

dark. Labeled protein spots in the gel were visualized using the Typhoon 9400 imager (GE

Healthcare). Gels were scanned using a 488 nm laser and an emission filter of 520 nm band pass

11

(BP) 40, a 532 nm laser and an emission filter of 580 nm BP30, a 633 nm laser and a 670 nm

BP30 emission filter to acquire the Cy2, Cy3, and Cy5 images, respectively. One of the four gels

was post-stained using Sypro Ruby (BioRad) accordingly to the manufacturer’s instructions,

visualized using the Typhoon 9400 scanner and then used as preparative gel for downstream

protein identification. In order to compare protein spots across the four gels, image analyses were

conducted in two steps using DeCyder v6.5 2D Differential Analysis Software (GE Healthcare).

Briefly, processed data were filtered to reveal statistically relevant fold changes in protein

abundance, using the average ratios of 5�1.5 and or 6�-1.5 fold differences in expression and one-

way ANOVA analysis with a p value of 6 0.05. Spots were excised from the gels with an

automatic spot picker (Investigator ProPic; Genomic Solutions Inc.), subjected to in-gel tryptic

digestion, concentrated with ZipTip® �37� ! !�##�� # !�� �� "" !���� ���� �"�#��� � ��)#"(� ��#�� ��

MALDI target. MALDI-MS and MALDI-MS/MS were performed on an Applied Biosystems

4800 plus MALDI TOF/TOF. The MS and MS/MS data were interpreted with the GPS Explorer

software (Version 3.6, Applied Biosystems). A combined MS peptide fingerprint and MS/MS

peptide sequencing search was performed against the NCBI database without taxon restriction

using the MASCOT search algorithm. These searches specified trypsin as the digestion enzyme,

carbamidomethylation of cysteine as fixed modification, partial oxidation of methionine and

phosphorylation of serine, threonine, and tyrosine as variable modifications, and allowed for one

missed trypsin cleavage. The monoisotopic precursor ion tolerance was set to 30 ppm and the

MS/MS ion tolerance to 0.3 Da. MS/MS peptide spectra with a minimum ion score confidence

interval 5�95% were accepted. This was equivalent to a median ion score cut off of approximately

35 in the data set. Protein identifications were accepted with a statistically significant MASCOT

protein search score 5� ��#%�#�)�����!������#�����������!��8�8 " #(��*�p < 0.05 in our data set.

12

Chromosomal spreading and multicolor-FISH. Karyograms were performed as previously

reported (41). In brief, parental CT26 cells, 1 hyperploid clone and 2 recovered tumors (from

immunodeficient and immunocompetent mice) were treated with 1 �M nocodazole overnight to

enrich mitotic cells, then collected and subjected to hypotonic lysis by incubation in 75 mM KCl

for 10 min at 37°C. After the removal of the hypotonic solution, cells were fixed in freshly

prepared Carnoy solution (3:1 methanol:acetic acid) and stored at -20°C. Fixed cells were then

dropped onto glass slides and hybridized with the 21XMouse Multicolor FISH (mFISH) Probe

Kit (MetaSystems, Altlussheim, Germany) following the manufacturer’s instructions. Briefly,

slides were denatured in 0.07 N NaOH and then rinsed in 70-100% ethanol series. Meanwhile,

the probe mix was denatured in a MJ mini personal thermal cycler (Bio-Rad laboratories,

Hercules, CA) with the following program: 5 min 75°C, 30 sec 10°C, and 30 min 37°C. Samples

were then hybridized in a humidified chamber at 37°C for 48 h followed by one wash in 1X

saline-sodium citrate (SSC) buffer for 5 min at 75°C and counterstaining with DAPI-containing

Vectashield®. Finally, metaphases were visualized and captured using an Axio-Imager M1

microscope equipped with six filter sets specific for the applied fluorochromes and with a charge

coupled device camera (Carl Zeiss, Jena, Germany). Karyotyping and cytogenetic analysis of

each single chromosome was performed by means of the ISIS software (MetaSystems). For each

experimental condition, at least 20 metaphases were analyzed. Chromosomes derived by the

fusion of the long arms of two different chromosomes in close proximity to the centromeric

region were approximated to a double chromosome in the calculation of the chromosome copy

number. Chromosome with ambiguous fluorochrome profiles were recorded as unknown. Of

note, in most cases, unknown chromosomes were essentially composed of centromeric DNA as a

side-result of fusion events.

13

Comparative genomic hybridization (CGH) array. DNA, extracted from 2 hyperploid CT26

clones and their corresponding tumors obtained from immunocompetent (BALB/c) and

immunodeficent (Rag �) mice, was evaluated using a Qubit spectrophotometer (Invitrogen). For

microarray hybridizations, DNA (500 ng) from each sample was digested and its integrity

measured using an Agilent BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). Test and

reference samples were labeled with the Cy5 and the Cy3 fluorescent dyes, respectively, by

means of the Genomic DNA Enzymatic Labeling Kit (Agilent Technologies), following the

manufacturer's protocol. Cy3-labeled and Cy5-labeled samples were then hybridized to the

SurePrint G3 Mouse CGH Microarray 4x180K (Agilent Technologies), prior to washing and

scanning with Agilent Scanner G2565CA. Oligonucleotide CGH processing was performed as

detailed in the manufacturer's protocol (version 4.0; http://www.agilent.com). Data were

extracted from scanned images using the Feature Extraction software v10.7.3 (Agilent

Technologies). Acquired signals were normalized according to dye and local GC% content using

in-house scripts under the R statistical environment (http://cran.r-project.org). Resulting log2

(ratio) were segmented using the CBS algorithm implementation from the R DNAcopy package

(42). Aberration status calling was automatically performed for each profile according to internal

noise (variation of log2 (ratio) values across consecutive probes on the genome). All genomic

coordinates were established on the UCSC Mus musculus genome build mm9 (43). Hierarchical

clustering was performed on the segmented data using Euclidean distances and the Ward’s

construction method. Differences across pairs of samples were quantified as the summed amount

in size of genome involved in gains and/or in losses for each comparison, and expressed as

percentage when compared to the size of the whole mouse genome.

14

Mice and genotyping. Mice were maintained in specific pathogen-free conditions, and

experiments followed the Federation of European Laboratory Animal Science Association

(FELASA) or National Health and Medical Research Council of Australia (NH&MRC)

guidelines. Animal experiments were in compliance with the EU Directive 63/2010 and were

approved by the Ethical Committee of the Institut Gustave Roussy (IGR, Villejuif, France)

(CEEA IRCIV/IGR n° 26, registered at the French Ministry of Research), the Peter MacCallum

Cancer Centre (PMCC, Melbourne, Australia) the University of Oviedo (Oviedo, Spain) or the

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (Vienna, Austria).

WT BALB/c and C57Bl/6 mice were obtained from Harlan France (Gannat, France), Janvier (Le

Genest St Isle, France) and Charles River Laboratories (Saint- Germain sur l’Arbresle, France);

Rag � and SCID/NOD mice from the IGR animal facility. Ifnar1-/- and Ifng-/- mice were kindly

provided by Dr. Matthew L. Albert (Institut Curie, Paris, France). To obtain primary Tp53-/- cells

, we cross-bred C57Bl/6 mice with a heterozygous deletion in the Tp53 gene (CNRS UMR6218,

Orleans, France), thereby generating Tp53+/+ (WT) and Tp53-/- animals. F1 mice were genotyped

by standard PCR procedures based on the following primers: p53-fwd (5'-

ACAGCGTGGTGGTACCTTAT-3'), p53-rev (5'-TATACTCAGAGCCGGCCT-3') and pNeo-

fwd (5'-CTATCAGGACATAGCGTTGG-3').

Antitumor vaccination and tumorigenicity assay. For vaccination experiments, 3x106 CT26

cells (be they WT or stably expressing ERp57- or CRT-targeting shRNAs) cells were left

untreated or treated with microtubule inhibitors for 48 h, and then were inoculated

subcutaneously in 200 µL PBS into the lower flank of 6-week-old female BALB/c mice. 5x105

untreated control cells were inoculated into the contralateral flank one week later. To restore CRT

at the plasma membrane of CT26 cells stably expressing a shRNA against CRT, before

15

inoculation, cells were incubated with recombinant CRT (generated in insect cells, 2-3 µg

recombinant protein/106 cells) in PBS on ice for 30 min, followed by three washes (39). For

tumorigenicity assays, 5x105 parental and hyperploid cells were injected subcutaneously into

BALB/c (class I MHC haplotype H-2d), C57Bl/6 (class I MHC haplotype H-2b) or Rag � (class I

MHC with the corresponding haplotype mice. Tumors were evaluated weekly using a common

caliper. Animals bearing tumors that exceeded 20–25% body mass were euthanatized. In some

experiments, BALB/c or C57Bl/6 mice that had previously been injected with hyperploid cells

but failed to develop tumors were re-injected with diploid cells, in order to establish the possible

vaccination quality of hyperploid cells. CD4+ and CD8+ lymphocytes were depleted in vivo by

the intraperitoneal injection of specific antibodies (see above) 5 days before tumor cell

inoculation, and on days 0, 1, 3, 5, 8, 15, 24 and 30. The depletion of CD4+ and CD8+ cells was

confirmed by immunofluorescence staining of spleen lymphocytes.

Priming assays. 3x106 (parental WT, hyperploid WT and hyperploid shCRT-transfected) CT26

cells were injected into the footpad of mice. Re-stimulation of draining popliteal node cells was

performed with heat-inactivated, dead CT26 cells (3x104 tumor cells heated for 5 min at 42°C,

followed by 1 cycle of freezing/thawing in liquid nitrogen). Supernatants were harvested 72 h

after re-stimulation and IFN� secretion was assessed by ELISA, as previously described (44).

Carcinogenesis models. Inbred WT Stat1-/-, and RAG-2 x �c-/- C57Bl/6 mice were bred and

maintained at the Peter MacCallum Cancer Centre. Dnam1+/+ E�-myc+ transgenic mice and their

Dnam1-/- E�-myc+ transgenic littermates (F2) were derived from breeding a Dnam1+/- E�-myc+

male with a Dnam1+/- E�-myc+ female (F1). This F1 generation was obtained by crossing a E�-

16

myc+ F0 male with a F0 Dnam1-/-. RAG-2 x �c-/- Il2-/- C57Bl/6 mice were purchased from The

Jackson Laboratory (Bar Harbor, ME, USA).

MCA model. WT and Stat1-/- male mice aged 6-10 weeks were injected subcutaneously in the

flank with 25 µg 3’-methylcholanthrene (MCA) dissolved in corn oil, as previously described

(32). Mice were then monitored 3 days a week for tumor growth over a period of 250 days. When

tumors reached 50-100 mm2, tumors and spleens were aseptically harvested, fixed in 10% neutral

buffered formalin and embedded in paraffin blocks.

E�-myc model. The F2 generation of Dnam1+/+ E�-myc+ mice and Dnam1-/- E�-myc+ mice was

monitored for the onset of spontaneous lymphomas. When mice showed lymphoma symptoms

such as an elevated white blood cell count, enlarged peripheral lymph nodes, lethargy, or troubled

breathing, the lymphoid tissue that manifested the disease (e.g., spleen, thymus, axillary/brachial

lymph nodes, inguinal lymph nodes, cervical lymph nodes) and peripheral blood were collected

and cell lines were generated. Additionally, some tissue was fixed in 10% neutral buffered

formalin and embedded in paraffin blocks.

DMBA/TPA model. For two-stage chemical carcinogenesis, the backs of 8-week-old-mice were

shaved and treated 2 days later with a single application of 7,12-� ��#%("8��9:�;��#%��)����

(DMBA, 25 µg in 200 µL acetone) followed by biweekly applications of 12-O-

tetradecanoylphorbol-13-acetate (TPA, 200 µL of 10-1 mM solution in acetone) for 12 weeks.

Mice were visually examined weekly and euthanatized if any individual tumor reached a

diameter of 2-3 mm or at the end of the experiment (16 weeks post-DMBA) (45). Tumors from

sacrificed animals were collected, fixed in 4% PFA and processed for histological studies.

17

MPA/DMBA model. RAG-2 x �c-/- Il2-/- C57Bl/6 mice were obtained from Taconic (Hudson,

NY, USA). Mammary tumors were induced by using medroxyprogesterone acetate (MPA) and

DMBA, as previously described (46-48). Briefly, 6 week old female mice were anesthetized

using Ketamine/Xylasol and received, as a subcutaneous implant, slow release

medroxyprogesterone acetate (MPA) pellets (50 mg/pellet, 90-days release, from Innovative

Research of America, Sarasota, FL, USA). In addition, mice received 1 mg DMBA (in 200 µL

corn oil) 6 times over a period of 8 weeks, according to a previously reported scheme (48). Mice

were routinely examined and mammary tumors were collected, upon mouse euthanasia, when

they reached a volume of 1 cm3. Collected tumors were fixed in 10% formalin overnight and

processed for histological studies.

Tumor patient selection. Breast cancer patients (Table S3) were treated with 3 cycles of

anthracycline and 3 cycles of docetaxel followed by surgery. “Responders” exhibited a complete

pathological response with only few residual cancer cells (Chevallier score 6� ,� <=�� -

responders” (Chevallier score 5����>% 8 #��� �?�� ?��!� ���(�#���������"(�!%��������#��#�����

(49).

Histology and immunohistochemistry. Samples from recovered tumors, mammary gland

tissues and human breast adenocarcinoma biopsies were fixed with 4% PFA for 4 h and then

embedded into paraffin. Sections of 10 �m were fixed and stained with haematoxylin and eosin

according to standard protocols. For phospho-�+���� �����% �#�)%�� �#�(��# �������)# ����$����

labeled with a monoclonal anti-phospho-�+��������� ��# 8��(� ��8��� �� /8)��� ��� ��# -

phospho-�+����������(3597, Cell signaling technology).

18

Supplemental References

36. M. Castedo et al., Selective resistance of tetraploid cancer cells against DNA damage-

induced apoptosis. Ann N Y Acad Sci 1090, 35-49 (2006).

37. L. Senovilla et al., p53 represses the polyploidization of primary mammary epithelial cells

by activating apoptosis. Cell Cycle 8, 1380-1385 (2009).

38. T. Sato et al., Long-term expansion of epithelial organoids from human colon, adenoma,

adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762-1772 (2011).

39. T. Sato et al., Single Lgr5 stem cells build crypt-villus structures in vitro without a

mesenchymal niche. Nature 459, 262-265 (2009).

40. T. Panaretakis et al., The co-translocation of ERp57 and calreticulin determines the

immunogenicity of cell death. Cell Death Differ 15, 1499-1509 (2008).

41. T. Iwawaki, R. Akai, K. Kohno, M. Miura, A transgenic mouse model for monitoring

endoplasmic reticulum stress. Nat Med 10, 98-102 (2004).

42. S. Mouhamad, L. Galluzzi, Y. Zermati, M. Castedo, G. Kroemer, Apaf-1 deficiency causes

chromosomal instability. Cell Cycle 6, 3103-3107 (2007).

43. A. B. Olshen, E. S. Venkatraman, R. Lucito, M. Wigler, Circular binary segmentation for

the analysis of array-based DNA copy number data. Biostatistics 5, 557-572 (2004).

44. D. Karolchik et al., The UCSC Genome Browser Database. Nucleic Acids Res 31, 51-54

(2003).

45. F. Ghiringhelli et al., Activation of the NLRP3 inflammasome in dendritic cells induces IL-

1beta-dependent adaptive immunity against tumors. Nat Med 15, 1170-1178 (2009).

46. M. Balbin et al., Loss of collagenase-2 confers increased skin tumor susceptibility to male

mice. Nat Genet 35, 252-257 (2003).

19

47. C. M. Aldaz, Q. Y. Liao, M. LaBate, D. A. Johnston, Medroxyprogesterone acetate

accelerates the development and increases the incidence of mouse mammary tumors

induced by dimethylbenzanthracene. Carcinogenesis 17, 2069-2072 (1996).

48. Y. Cao, J. L. Luo, M. Karin, IkappaB kinase alpha kinase activity is required for self-

renewal of ErbB2/Her2-transformed mammary tumor-initiating cells. Proc Natl Acad Sci U

S A 104, 15852-15857 (2007).

49. D. Schramek et al., Osteoclast differentiation factor RANKL controls development of

progestin-driven mammary cancer. Nature 468, 98-102 (2010).

50. B. Chevallier et al., A prognostic score in histological node negative breast cancer. Br J

Cancer 61, 436-440 (1990).

51. C. P. Yang et al., A highly epothilone B-resistant A549 cell line with mutations in tubulin

that confer drug dependence. Mol Cancer Ther 4, 987-995 (2005).

20

Legends to Supplementary Items

Figure S1: CRT exposure in response to tetraploidizing agents, as detected by different

techniques. A-D, Identification of tetraploidizing agents as CRT-exposing agents in human

osteosarcoma U2OS cells stably co-expressing CRT-GFP and H2B-RFP. A, Screening of the

ICCB library (including 480 bioactive compounds) for the identification of agents that induce the

redistribution of CRT into peripheral dots (“CRT granularity”). B, Kinetics of nocodazole-

induced redistribution of CRT-GFP to the cell periphery, as assessed by videomicroscopy.

Representative pictures and quantitative data (at 4 h) are shown in C and D, respectively. Scale

bar, 10 µm. Experiments were performed three times, yielding similar results. Samples were

compared using one-tailed Student’s t test. Error bars indicate SEM. *p<0.05, **p<0.01,

compared to untreated cells. E-H, Assessment of CRT exposure in response to microtubular

inhibitors by means of the HaloTag® system. Human osteosarcoma U2OS cells stably transfected

with a HaloTag®-CRT (HT-CRT)-expressing construct (E) were treated for the indicated time

$ #%� �� ��� ? �)� �# ��� �@3�� ,�� ��� )(#�)%�"�� �� �� �3(#��� A� ��� ? �8"��# ��� �@'�� A� ���

)�")% ) ����33��0����)(#�)%�"�� ��B��3(#B���������!�)" #�>�"�(PCT) or 100 nM nocodazole

(Noco), followed by FACS-assisted detection of HT-CRT exposure after staining with a cell-

impermeable HaloTag® Alexa Fluor® 488 ligand. Representative FACS pictogrames (at 24 h)

are shown in F, quantitative data in G and H. Experiments were performed three times, yielding

similar results. Samples were compared using one-tailed Student’s t test. Error bars indicate

SEM. *p<0.05, **p<0.01, compared to untreated cells. I-J, Cytofluorometric assessment of CRT

exposure in response to microtubular inhibitors. Human osteosarcoma U2OS cells were treated

for the indicated time with microtubular inhibitors as in G-H and then subjected to

immunostaining with a CRT-specific antibody followed by FACS analysis. Representative

21

pictograms (at 24 h) are shown in I, quantitative data in J. Experiments were performed three

times, yielding similar results. Samples were compared using one-tailed Student’s t test. Error

bars indicate SEM. *p<0.05, **p<0.01, compared to untreated cells.

Figure S2: CRT exposure is a cell-autonomous event. Cell-autonomous origin of cell-surface

CRT on cytochalasin D (CytD)-treated cells. A, CRT exposure on viable cells. Human colon

carcinoma HCT 116 cells were either left untreated (control) or cultured for 48 h in the presence

of ,�� ��� cytochalasin D (CytD), followed by immunostaining for the detection of surface-

exposed CRT (ecto-CRT) plus counterstaining with the vital dye propidium iodide (PI), and

cytofluorometric analysis. Side (SSC) and forward scatters (FSC) were used to eliminate debris

from data analysis, and PI positivity to exclude dead cells from the analysis. Finally, cells

belonging to both gate 1 and 2 were assessed for CRT surface expression. B, Failure of CRT

from dying or dead cells to bind to live cells. Wild type (WT) HCT 116 cells were cultured in the

absence or presence of 1.2 µM CytD for 48 h, washed and recultured for further 24 h, followed

by the detection of surface CRT by indirect immunofluorescence. The same study was performed

on H2B-GFP-expressing HCT 116 cells that were kept in control conditions for 48 h or co-

incubated with WT HCT 116 cells that had been pre-treated with CytD for 48 h. The ratio of

H2B-GFP-expressing to WT HCT 116 cells was 10:1. Note the absence of CRT transfer from

CytD-treated WT to H2B-GFP-expressing HCT 116 cells. C, Failure of the HaloTag-CRT fusion

protein to shuttle from dying or dead to live cells. Human osteosarcoma U2OS cells stably

expressing HaloTag-CRT fusion protein (HT-CRT) were split into two aliquots, one that was

either treated with 1.2 µM CytD for 48 h, followed by three washes, and 24 h of culture in drug-

free conditions or left untreated for the same time, and another one that was labeled with

MitoTracker® Deep Red FM (Molecular Probes), following the manufacturer’s instructions.

22

These latter cells were left cultured or not in the presence of unlabelled, CytD-pretreated.

Surface-exposed HaloTag-CRT was detected by means of an impermeant HaloTag ligand

(HaloTag ligand Alexa 488) before cytofluorometric analysis. Numbers in B and C indicate the

percentage of cells found in each quadrant. Results are representative of three independent

experiments yielding similar results.

Figure S3: CRT exposure in different models of tetraploidization. A-G, Exposure of CRT

upon acute exposure to multiple microtubular inhibitors in a variety of murine and human cancer

cell lines, as well as in Tp53-/- primary mouse intestinal cells (IECs) and mammary gland

epithelial cells (MGCs). Murine colon carcinoma CT26 cells (n=4, A), human colon carcinoma

HCT 116 cells (n=5, B), or human wild type non-small cell lung cancer A549 cells (n=7, C) were

treated with 30 nM epothilone B (EpoB), 100 nM taxotere (TXT) or 300 nM paclitaxel (PCT)

overnight. A549-derived B480 cells (n=10, C), that had been selected in – and are addicted to the

continuous presence of – epothilone B (due to several tubulin � and � mutations) (50), were

allowed to tetraploidize by the removal of epothilone B. Alternatively, HCT 116 cells (n=4, D),

murine Lewis lung cancer (LLC) cells (n=3, E), IECs (n=3, F) and MGCs (n=14, G) were treated

with 1.2 �M cytochalasin D (CytD), 2 µM dihydrocytochalasin B (DCB) or 100 nM nocodazole

(Noco) overnight. Finally, CRT exposure was determined by cytofluorometry. Representative

immunofluorescence pictures are shown in A, and quantitative data in B-G. Scale bar, 5 µm. H,

Exposure of CRT by inhibition of the spindle assembly checkpoint. HCT 116 cells were

transfected with a control siRNA (siUNR) or with siRNAs targeting Polo-like kinase 1 (siPLK1),

MAD2 (siMAD2) or Aurora kinase B (siAURKB) for 72 h, followed by the cytofluorometric

assessment of ploidy and CRT exposure. Quantitative data are reported. I, CRT exposure on

spontaneous hyperploid clones. Human colorectal carcinoma RKO cells stably expressing an

23

histone 2B-GFP (H2B-GFP) fusion and Hoechst 33342-labeled LLC cells were sorted according

to DNA content and allowed to generate clones for 14 days. Thereafter, stable parental and

hyperploid clones were characterized for CRT exposure by cytofluorometry. Quantitative data

are reported (n=4 clones per condition). J, CRT exposure on chemically-induced hyperploid

clones. CT26 cells were treated with 1.2 �M CytD for 48 h, FACS-sorted according to DNA

content (upon Hoechst 33342 staining), and allowed to generate clones for 14 days. Thereafter,

CRT exposure was assessed in stable clones as in I. Quantitative data are reported (n=5-6 clones

per condition). Results were normalized to baseline ecto-CRT levels as observed in non-treated

CT26 cells. K. CRT exposure on hyperploid colon crypt organoids. Diploid and tetraploid

organoids were derived from FACS-sorted Tp53-/- colon epithelial stem cells and assessed for

CRT exposure by immunofluorescence microscopy. Representative images and quantitative data

are shown (scale bar, 100 �m). Samples were compared using one-tailed Student’s t test. Error

bars indicate SEM. *p<0.05, **p <0.01, compared to untreated cells.

Figure S4: CRT exposure on non-transformed and malignant hyperploid, but not trisomic,

cells. hTERT-immortalized human foreskin BJ5ta fibroblasts were treated with 1.2 �M

cytochalasin D for 48 h, sorted by cytofluorometry (upon Hoechst 33342 staining) according to

DNA content and allowed to generate stable diploid and tetraploid clones. Clones were then

characterized for DNA content by cytofluorometry (A), ploidy by interphase FISH on

chromosomes 2 (red), 8 (green) and 10 (white) (in > 100 cells per condition) (B), CRT exposure

by cytofluorometry (C�� ���� !%��!%��("�# ��� "�?�"�� �*� �+���� 8(� �����8"�## ��� )��!"��� #��

densitometric image analysis (D). Representative results are illustrated in A,B and D. Scale bar, 5

��,� C���# #�# ?�� ��#�� ���� ��!��#��� �� C and D. Samples were compared using one-tailed

Student’s t test. Error bars indicate SEM. *p<0.05, compared to diploid cells. E,F, Trisomy does

24

not suffice to induce CRT exposure. Human colon carcinoma DLD-1 cells and their trisomic

counterparts DLD-1+7 (which bear an additional copy of chromosome 7) (25) were characterized

for CRT exposure by cytofluorometry (E� ���� !%��!%��("�# ��� "�?�"�� �*� �+���� 8(�

immunoblotting (F). As a positive control for hyperploidization-induced CRT exposure, DLD-1

and DLD-1+7 cells were treated with 1.2 �M cytochalasin D for 48 h, sorted by cytofluorometry

(upon Hoechst 33342 staining) according to DNA content, cultured as polyclonal populations for

12-14 days, and then monitored for CRT exposure (E������+����!%��!%��("�# ����F). Samples

were compared using one-tailed Student’s t test. Error bars indicate SEM. *p<0.05, **p<0.01,

compared to parental cells (B-D) or to untreated cells (E).

Figure S5: Manifestations of ER stress in cells treated with microtubular inhibitors. Wild

type human osteosarcoma U2OS cells (A,B) or U2OS stably expressing a XBP1-DBD-Venus- or

GFP-ATF6-encoding construct (C-D) were either left untreated or incubated for the indicated

t ���$ #%�����? �8"��# ����@'������? �)� �# ����@3������!���* "�>��2�������!�)" #�>�"�

(PCT), 500 nM thapsigargin (Thap, an inhibitor of the SERCA pump of the ER, used as positive

)��#��"�*����D��#����� ���)# ������������)���9�"���=�)���,�����)(#�chalasin D (CytD), 30

��� �!�#% "���� 8� ��!�'�� ��� ��� #�>�#���� �.E.� ��� �,0� ��� 8��#�9�� 8� �'.F�� �� !��#�������

inhibitor, used as positive control). Thereafter, cells were processed for the software-assisted,

(immuno)fluorescence-based detection of phosphoryl�#��� �+���� �A), XBP1 expression (C) and

ATF6 perinuclear translocation (E,F,� /"#����# ?�"(�� !%��!%��("�#��� ���� #�#�"� �+���� $����

assessed by quantitative immunoblotting (B), and XBP1 expression by cytofluorometry (D).

Representative images and single cell profiles (at 15 h) are reported in A, C and E, quantitative

data in B (at 48 h), D and F. Samples were compared using one-tailed Student’s t test. Error bars

indicate SEM. *p<0.05, **p <0.01, compared to untreated cells.

25

Figure S6: Signals involved in nocodazole-induced CRT exposure. Mouse embryonic

fibroblasts (MEFs) with the indicated genotypes (A) or mouse colon carcinoma CT26 cells stably

expressing different shRNAs (B) were treated with nocodazole for 48 h and CRT exposure was

measured by immunofluorescence. Samples were compared using one-tailed Student’s t test.

Error bars indicate SEM. *p<0.05, **p <0.01, compared to wild type (WT) MEFs (A) or CT26

cells transfected with a control shRNA (shCo) (B).

Figure S7: Immunogenicity of cells treated with microtubular inhibitors. BALB/c mice were

injected s.c. with wild type (WT) murine colon carcinoma CT26 cells that had been treated or not

with 30 nM epothilone B for 48 h, or with CT26 cells stably transfected with an ERp57-specific

shRNA (shERp57) and treated with epothilone B as above. Mice were re-challenged one week

later with live WT CT26 cells and tumor incidence was monitored. Experiments were performed

three times (15-20 mice per group). Survival curves were compared using the Log rank test. Error

bars indicate SEM. *p<0.05, **p <0.01, compared to mice challenged with untreated WT CT26

cells.

Figure S8: Increase in surface area does not account for increased CRT exposure on

hyperploid cells. A-C, Two-dimensional gel electrophoresis of purified plasma membrane

proteins from parental and hyperploid murine colon carcinoma CT26 cells. After cell surface

biotinylation, biotinylated (and hence cell surface-exposed) proteins were purified and subjected

to two-dimensional gel electrophoresis, computer-aided measurements of spot intensities and

mass spectrometric identification. Representative gels are shown in A. The list in B indicates the

ratio obtained by the comparison between parental (n=3) and hyperploid (n=3) clones. Positive

and negative values indicate up- and downregulation, respectively. Panel C, depicts spots

corresponding to ecto-CRT in two-dimensional gels. D, Immunodetection of other surface

26

proteins such as the NKG2D ligand Rae-1 (n=10), the death receptor CD95/Fas (n=6), and the

adherence receptor CD47 (n=6) in parental and hyperploid CT26 clones. Samples were compared

using one-tailed Student’s t test. Error bars indicate SEM. *p<0.05, **p<0.01, compared to

parental CT 26 cells.

Figure S9: Enhanced exposure of stress molecules at the surface of stable hyperploid clones.

A-D, Enhanced exposure of stress molecules at the surface of stable hyperploid clones. A,

Features of hyperploid clones. The table summarizes the number, ploidy and CRT exposure of

the near-to-tetraploid clones used in this study, and the drugs used for their generation. Please

note that even spontaneous near-to-tetraploid clones exhibited enhanced CRT exposure. Samples

were compared using one-tailed Student’s t test. Error bars indicate SEM. *p<0.05, **p <0.01. B,

Parental and hyperploid clones generated from murine fibrosarcoma MCA205 (n=5) and Lewis

lung carcinoma LLC (n=12) were subjected to immunofluorescence for the detection of surface

CRT. C, Parental and hyperploid clones generated from murine colon carcinoma CT26 (n=5),

murine Lewis lung carcinoma LLC (n=12) and human wild type (WT) colon carcinoma HCT 116

(n=3) cells were subjected to immunofluorescence for the detection of externalized ERp57. D,

WT (n=11) and TP53-/- (n=14) near-to-diploid and near-to-tetraploid HCT 116 clones were

compared for basal CRT exposure.

Figure S10: Immunosurveillance elicited by hyperploid cancer cells. A-B. Tumor growth

assay. Parental (P) or hyperploid (H) clones from murine Lewis lung carcinoma LLC (A) and

murine fibrosarcoma MCA205 (B) cells were inoculated in Rag � or C57Bl/6 mice, and tumor

growth (upper panels) and incidence (lower panels) were monitored. Error bars indicate SEM.

Tumor incidences (illustrated with Kaplan-Meier curves) were compared by the Log rank test.

*p<0.05, **p<0.01, compared to Parental inoculated mice. C. Correlation between T cell priming

27

and ecto-CRT in hyperploid tumor cells. Hyperploid (H) or parental (P) wild type (WT) murine

colon carcinoma CT26 cells or hyperploid CT26 cells stably expressing an shRNA for the

downregulation of CRT (shCRT) were assessed for spontaneous CRT exposure by

immunofluorescence or injected into the footpads of BALB/c mice. Five days later, draining

popliteal lymph node cells were restimulated with parental tumor lysates, and IFNG production

was measured 72 h later. Error bars indicate SEM. Samples were compared with one-tailed

Student’s t test. **p<0.01, compared to parental cells; ##p<0.01, compared to hyperploid WT

cells.

Figure S11: Immunogenicity of hyperploid cells. Immunocompetent C57Bl/6 mice were

inoculated with PBS or near-to-tetraploid murine fibrosarcoma MCA205 cells (A). PBS-injected

mice as well as those mice that stayed tumor free for one month (approximately 40% of all

inoculated mice) were reinjected with either parental MCA205 (B) cells or with unrelated murine

Lewis lung carcinoma LLC (C) cells. Thereafter, tumor growth and incidence were routinely

monitored, on both control (n=8) and challenged (n=10) mice. Tumor incidences (illustrated with

Kaplan-Meier curves) were compared by the Log rank test. *p <0.05, **p <0.01, compared to

parental cells.

Figure S12: Altered ploidy in CT26 cells undergoing in vivo immunoselection. A-C.

Representative hyperploid CT26 tumors showing reduced nuclear diameter, as determined by

hematoxylin/eosin staining (A; scale bar, 10 ��), decreased chromosome numbers, as assessed

on metaphase spreads (B; scale bar, �� ��) and lowered DNA content, as assessed by

cytofluorometry (C), upon immunoselection in immunocompetent (BALB/c), but not from

immunodeficient (Rag �), mice. Quantitative data are reported in Figure 2A-C.

28

Figure S13: CT26 karyotype by multicolor FISH. Multicolor FISH of tumors derived from

murine colon carcinoma CT26 parental cells and one near-to-tetraploid CT26 clones. A,

Representative images (upper panels) and the corresponding reordered karyotypes (lower panels).

B, Numbers of chromosome copies of parental CT26 cells, the hyperploid clone and two tumors

generated from this clone, one in immunodeficient (ID) Rag � mice and the other in

immunocompetent (IC) BALB/c mice. U means unknown chromosome. Karyotypes were

determined for 20 cells in each case, and results are given as means±SEM. Data were compared

with one-tailed Student’s t test. The copy number of all chromosomes was significantly different

(p<0.01) in hyperploid versus parental CT26 cells. *p<0.05, **p<0.01, compared to the

hyperploid clone; #p<0.05, ##p<0.01, compared the tumor recovered from ID mice.

Figure S14: Immunoselection reduces ploidy and the exposure of stress molecules at the cell

surface. A,B. DNA loss from immunoselected hyperploids. Hyperploid clones from murine

Lewis lung carcinoma LLC (A) and murine fibrosarcoma MCA205 (B) cells were cultured in

vitro, immunoselected in C57Bl/6 mice or grown in vivo without immunoselection in Rag � mice,

recovered before tumor volume exceeded 3 cm3 and their ploidy was determined by

cytofluorometry. C-E. Immunoselection affects CRT/ERp57 exposure. Hyperploid clones from

murine Lewis lung carcinoma LLC (C) and murine fibrosarcoma MCA205 (D) cells were either

cultured in vitro, grown in vivo without immunoselection (in Rag � mice), or grown on

immunocompetent (C57Bl/6) mice for immunoselection, recovered no less than one month later

and then assessed for CRT exposure by cytofluorometry. Alternatively, colon carcinoma CT26

hyperploid clones cultured in vitro or grown in vivo on Rag � or BALB/c mice for at least one

week were characterized for ERp57 exposure by cytofluorometry (E). One-tailed Student’s t test

29

was used for statistical comparisons. Error bars indicate SEM. *p<0.05, **p<0.01, compared to

cells cultured in vitro.

Figure S15: Reduced ploidy and ER stress after immunoselection. A-D, Representative

pictures of methylcholanthrene-induced fibrosarcomas (A,B), E�-myc-driven B cell lymphomas

(C), or medroxyprogesterone acetate-induced mammary carcinomas (D) from immunodeficient

�D���G��Stat1-/- or Dnam1-/-) versus immunocompetent mice, upon immunohistochemical staining

for the detections of eIF2� phosphorylation. Scale bar, 10 �m. E, Representative pictures of

mammary carcinomas from patients who did (responders) or did not (non-responders) respond to

chemotherapy, upon immunohistochemical staining for the detection of eIF2� phosphorylation

(upper panels), CD8+ lymphocytes (middle panels) and FOXP3+ lymphocytes (lower panels).

Figure S1

CR

T ga

nula

rity

1.4

1.2

1.0

0.8

ICCB Compounds

95% percentile

Calycu

lin A

Cytoch

alasin

D

Nocod

azole

Paclita

xel

Vinblas

tine

A B

Time after nocodazole addition

CRT-GFP

H2B-RFP 0h 3h 6h

Paclitaxel

Calyculin A

Nocodazole

CRT-GFPH2B-RFP

Control Cytochalasin D

Vinblastine

C D

Vinblas

tine

Paclita

xel

Nocod

azole

Cytoch

alasin

D

Calycu

lin A

Contro

l

GFP

-CR

T gr

anul

arity

1.4

1.2

1.0

0.8

**

****

010203040506070

0 101 102 103 1040

10203040506070 Control

Vinblastine

ControlCytD

0 101 102 103 104

ControlNocodazole

ControlVincristine

0 101 102 103 104

ControlPaclitaxel

Ecto-CRT

IsotypeControl

I

Cou

nts

E G

H

** ** * ** **

0

1

2

3

4

524h4h

HT-

CR

T (A

.U.)

F

HT-CRT

ControlVincristine

0 101 102 103

ControlPaclitaxel

ControlCytD

0 101 102 103 104

ControlNocodazoleC

ount

s

01020304050

0 101 102 103 1040

1020304050 Control

Vinblastine

No LigandUntreated

control+ Ligand

104 Co

HaloTag®CRT KDEL

pCDNA3

0 1 2 4 6 17 24 48

Nocodazole

**

012345

HT-

CR

T (A

.U.)

Time (h)

****

**V

CC

ytD VB

CC

Cyt

HP

CT

Noc

oC

oV

CC

ytD VB

CC

Cyt

HP

CT

Noc

o

** ** ***

*

*

0

1

2

3

4

Ect

o-C

RT

(R.U

.)

Co

VC

Cyt

D VB

CC

Cyt

HP

CT

Noc

oC

oV

CC

ytD VB

CC

Cyt

HP

CT

Noc

o

J24h4h

A

B

GFP-H2B

FSC (x102)

SS

CControl isotype Control CRT CytD isotype CytD CRT

100

104

103

102

101

20 4 6 8 10 20 4 6 8 10 20 4 6 8 10 20 4 6 8 10

Gate 1 Gate 1 Gate 1 Gate 1

PI

100

104

103

102

101

100 104103102101 100 104103102101 100 104103102101 100 104103102101

Gate 2 Gate 2 Gate 2 Gate 2

Ecto-CRT

Ecto-CRT

Cou

nts

(Gat

e1 +

Gat

e 2)

100 104103102101 100 104103102101 100 104103102101 100 1041031021010

5040302010

Ect

o-C

RT

WT control WT CytDGFP-H2B controlGFP-H2B control

WT CytD

100

103

102

101

100 103102101 100 103102101 100 103102101 100 103102101

0.0 0.0

0.199.9

0.1 2.2

97.00.6

18.0 0.0

0.082.0

15.2 1.0

11.772.0

C

MitoTracker® Deep Red FM

HT-

CR

T

100 103102101 100 103102101 100 103102101 100 103102101

100

103

102

101

0.8 0.0

0.099.2

0.0 1.7

98.10.2

12.6 0.0

0.087.4

14.4 0.5

19.365.8

HT control HT Control-MitoTracker HT CytDHT Control-MitoTracker

HT CytD

Figure S2

Figure S3

ACT26

CHCT 116

B

D

Contro

lCytD Noc

o

IECsFE

DCB

GCon

trol

EpoB

TXTPCT

Contro

lNoc

o

Contro

lNoc

o

0

1

2

3

****

*

Ect

o-C

RT

(R.U

.)

MGCs

0

2

4

5

1

3**

DCB

Contro

l

Ect

o-C

RT

(R.U

.)

0

1

2 *

Ect

o-C

RT

(R.U

.)

LLC

02

46

8

10*

*

Ect

o-C

RT

(R.U

.)

**

0

2

4

6HCT 116

**

Ect

o-C

RT

(R.U

.)A549

WT B480(n=7) (n=10)

0

2

4

6

8

10 -EpoB+EpoB

**

*

Ect

o-C

RT

(R.U

.)HoechstCRT

Control EpoB

% of >4n cells

Ect

o-C

RT

(R.U

.)

0 10 20 30 400

1

2

3

siUNR

siAURKB

siPLK1siMAD2

J

0.0

0.5

1.5

1.0

2.0E

cto-

CR

T (R

.U.)

**

**# #

K

Hoechst CRT Bright Field

Dip

loid

Tetra

ploi

d

Diploid Tetraploid0

1

3

2

4

Ect

o-C

RT

(R.U

.)

5**

Parenta

l

Hyperp

loid

0.0

0.5

1.5

1.0

2.0

Ect

o-C

RT

(R.U

.) **

RKO H2B-GFP

**

0

2

6

4

8

Ect

o-C

RT

(R.U

.)

I

Parenta

l

Hyperp

loid

LLCHCT 116H

CT26

Parenta

l

Parenta

l

Hyperp

loid

WTClones

Figure S4

Diploid TetraploidTetraploid

2n4n 8n

Diploid

2n4n 8n

Cou

nts

A B

P-eIF2α

eIF2α 38 KDa

38 KDa

Diploid

Tetra

ploid

*

0

1

2

4

eIF2

α (P

/T, R

.U.)

Diploid Tetraploid

3

D

0

1

2

3

Ect

o-C

RT

(R.U

)

Diploid Tetraploid

*C

Control Cyt D0

2

4

6

8

10

Ect

o-C

RT

(R.U

)

DLD-1DLD-1+7 **

**

E F

P-eIF2α

eIF2α 38 KDa

38 KDa

DLD-1

DLD-1+

7DLD

-1

DLD-1+

7

Control CytD

Noco CytD EpoB TXT

C 60

40

20

0∗∗∗

∗∗

∗∗

∗∗

∗∗∗∗

∗∗

∗∗∗∗

HoechstGFP-XBP1 Control

Log

(Inte

nsity

of

GFP

-XB

P1

per c

ell)

2.11

2.44

2.78

3.11

3.44VB VC PD PCT Thap

20% 96% 95% 92% 94% 95%

80% 4% 5% 8% 6% 5%

2 4 6 2 4 6 2 4 6 2 4 6 2 4 6 2 4 6Log (Mean nuclear size)

XB

P1 +

cells

(%)

0 6 24 48 (h)6 24 48 6 24 48 6 24 48

D

E

HoechstGFP-ATF6

Log

(Inte

nsity

of

GFP

-ATF

6 pe

r cel

l)

2.11

2.44

2.78

3.11

3.44

2 4 6 2 4 6 2 4 6 2 4 6 2 4 6 2 4 6Log (Mean nuclear size)

Control VB VC PD PCT BTZ

14% 91% 90% 96% 70% 92%

86% 9% 10% 4% 30% 8%

100

40

20

0ATF6

tran

sloc

atio

n (%

of c

ells

)

60

80

F

0 24 48

Noco

CytDEpo

BTXT

(h)24 48 24 48 24 4824 48

Tun

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

BP-eIF2α

eIF2α38 kDa

Con

trol

∗

Cyt

D

∗∗

Noc

o

∗

Epo

B

∗

TXT

∗∗

Thap

0

1

2

3

4

eIF2

-α (P

/T)

Control VB VC PD PCT ThapHoechstP-eIF2α

A

2 4 6 2 4 6 2 4 6 2 4 6 2 4 6 2 4 6Log (Mean nuclear size)

Log

(Inte

nsity

of

P-e

IF2α

per

cel

l)

2.23

2.54

2.85

3.15

3.4610% 98% 92% 91% 86% 96%

90% 2% 8% 9% 14% 4%

38 kDa

Figure S5

Figure S6

A 8

6

4

2

0

B

Bax-/- B

ak-/-

Casp8

-/-

WT

eIF2α

S51A

Nocodazole

*

**

6

4

2

0

Nocodazole

shCosh

PERK

shCas

p8

Ect

o-C

RT

(R.U

.)

Ect

o-C

RT

(R.U

.)

shERp5

7

shCRT

* *

** **

Figure S7

0 10 20 30Time (d)

0

20

40

60

80

100

Tum

our-

free

mic

e (%

)

Day (-7) Day (0)

Live WT CT26Monitor

tumor growth

Challenge

**

PBS

CT26 shERp57 Epothilone B

n.s.

WT CT26 Epothilone B

A

UPREGULATED IN HYPERPLOID CELLS: 19 SPOTS CORRESPONDING TO 11 PROTEINSNº Protein Name Ratio p value

60 kDa heat shock protein, mitochondrial (chaperonin) [Mus musculus]427 2 0.01430 60 kDa heat shock protein, mitochondrial [Mus musculus] 1.8 0.01418433

60 kDa heat shock protein, mitochondrial [Mus musculus]60 kDa heat shock protein, mitochondrial [Mus musculus]

1.52

0.030.02

408 calreticulin [Mus musculus] 2 0.051328 eukaryotic translation initiation factor 1A [Mus musculus] 1.8 0.0021071 G protein beta subunit like [Mus musculus] 1.9 0.00031083 G protein beta subunit like [Mus musculus] 1.9 6.8 e-0051136 galectin-3 [Mus musculus] 2.3 1.8 e-0051140 galectin-3 [Mus musculus] 2 6.9 e-005755 histocompatibility 2, D region locus 1 [Mus musculus] 2.4 0.02728 histocompatibility 2, D region locus 1 [Mus musculus]727 histocompatibility 2, D region locus 1 [Mus musculus] 2

2.3 0.010.001

885 laminin receptor 1 (ribosomal protein SA) [Mus musculus] 2.5 0.001913 laminin receptor 1 (ribosomal protein SA) [Mus musculus] 2 2.6 e-005489 nucleosome assembly protein 1-like 1 [Mus musculus] 1.7 0.001

1198 peroxiredoxin 6 [Mus musculus] 1.8 0.03279 1.8stress-70 protein, mitochondrial [Mus musculus] 0.002923 translocase of inner mitochondrial membrane 50 homolog [Mus musculus] 1.7 0.003

DOWNREGULATED IN HYPERPLOID CELLS: 9 SPOTS CORRESPONDING TO 7 PROTEINSNº Protein Name Ratio p value

924 Anxa1 protein [Mus musculus] -1.8 0.005684 elongation factor 1-gamma [Mus musculus] -1.6 0.002386 elongation factor 1-gamma [Mus musculus] -1.5 0.003377 prolyl 4-hydroxylase subunit alpha-1 [Mus musculus] -2 0.01346 protein methyltransferase [Mus musculus] -1.7 0.003485 pyruvate kinase 3 [Mus musculus] -1.5 0.005488 pyruvate kinase 3 [Mus musculus] -2.1 0.01385 Scfd1 protein [Mus musculus] -1.8 0.001514 vimentin [Mus musculus] -2.8 0.01

408

489

885913

1328

1198

514433

427

430418

279377 386385346

684727

755

728

923 924

488485

114011361071

1083

408

489

408

885913

1198

1328

114011361071

1083

923 924

684727

755

728

488485

386385346377279

418514433

427

430

pH3 - 10 NL pH3 - 10 NL

10075

50

37

25

20

KDa

Parental Hyperploid

489

Upregulated proteinDownregulated protein

B

Exp

osed

pro

tein

s (R

.U.)

Rae-1 CD95/Fas CD47

D

0.0

0.2

0.4

0.6

0.8

1.0

1.2 ParentalHyperploid

** **

Figure S8pH 3 - 4 NL

75

50KDa pH 3 - 4 NL

Parental HyperploidC

Figure S9

A

0

2

4

6

8

10

Ect

o-E

Rp5

7 (R

.U.)

C

WT HCT 116

LLCCT26

**

*

**

D

WT HCT 116

TP53-/-

HCT 116

0

1

2

3

Ect

o-C

RT

(R.U

.) ***

Cou

nts

Ecto-CRT 0

2

4

6

8

10

MCA205

LLC

n=12

**n=5

**

Ect

o-C

RT

(R.U

.)

BParentalHyperploid

Cell line

Hyperploidizing Agent

Number of hyperploid clones

Ploidy of hyperploid clones (diploid=2) ± SEM

CRT exposure (parental controls=1) ± SEM

RKO H2B-GFP None 3 3.7 ± 0.02 ** 1.5 ± 0.1 ** CT26 Nocodazole 34 3.8 ± 0.9 ** 2.4 ± 0.5 ** CT26 Cytochalasin D 5 3.8 ± 0.4** 2.0 ± 0.1 ** MCA 205 Nocodazole 3 4.1 ± 0.04 ** 2.2 ± 0.3 ** LLC None 3 4.2 ± 0.04** 6.0 ± 0.7 ** LLC Dihydro-cytochalasin B 3 4.8 ± 0.1 ** 6.6 ± 1.8 ** HCT 116 Cytochalasin D 3 3.8 ± 0.3 * 1.8 ± 0.4 * Bj5ta Cytochalasin D 1 4 2.2 ± 0.5 *

Figure S10

B MCA205

Time (d)0 10 20 30 40

020406080

100

Tum

our-

free

mic

e (%

)Tu

mou

r vol

. (cm

3 )

Rag γ

HyperploidParental

01234 (n=20)

C57Bl/6

***

****

(n=20)

0 10 20 30 40

*

A

Time (d)

LLC

020406080

100

Tum

our-

free

mic

e (%

)

100 20 30

Tum

our v

ol. (

cm3 )

01234

Rag γ

(n=10)HyperploidParental

100 20 30

**

C57Bl/6

** ** **

(n=20)

1

0

2

P H H

WT

shCRT

IFNγ (ng/m

L)1

0

3

2

Ect

o-C

RT

(R.U

.)

**

**

##

##

C

PBS

MCA205Hyperploid

1 month

MCA205Parental

LLCParentalor

Control

Challenged

A

B

0

1

2

3

4

Tum

our V

ol. (

cm3 ) Control (n=10)

Challenged (n=8)

MCA205

** ** ** ** ** **0 5 1510 20 3025 35

Time (d)

0

20

40

60

80

100

Tum

our-

free

mic

e (%

)

0 5 1510 20 3025 35Time (d)

**

100 20Time (d)

Control (n=10)Challenged (n=8)

LLC

0.0

0.5

1.0

1.5

2.0

Tum

our V

ol. (

cm3 )

5 15 25

C

0

20

40

60

80

100

Tum

our-

free

mic

e (%

)

100 20Time (d)

5 15 25

Figure S11

Figure S12

BALB/cRag γA

Rag γ

148 75

BALB/cB

Cou

nts

2n 4n 8n 2n 4n 8n

BALB/cRag γIn vitroIn vivo

C

Figure S13

AParental CT26 Hyperploid clone

BParental

Num

ber o

f cop

ies

0

2

4

6

8

10 ID Tumor IC Tumor

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X UChromosome

Hyperploid

** ** ** **

**

**** **

*

** **

** **

***

## ##

##

##

***

##

#

##

****

#

****

##

***

##

**##

**##

Figure S14

**

3

4

MCA205LLC

C57Bl/6

n=7

*

n=4

*

n=4

n=6

In vit

roRag

γ

In vit

roRag

γ

C57Bl/6

2

3

4P

loid

y

A

Plo

idy

B

n=4 n=4

C57Bl/6

*

*

MCA205(n=6)

In vit

roRag

γ

**

LLC

0.0

0.5

1.0

1.5 (n=12)

In vit

roRag

γ

C57Bl/6

Ect

o-C

RT

(R.U

.)

C

**

0

1

2

3

4

5

Ect

o-E

Rp5

7 (R

.U.)

BALB/c

In vit

roRag

γ

ECT26

n=7*

n=8

0.0

0.5

1.0

1.5

Ect

o-C

RT

(R.U

.)

D

n=8

A

B

C

C57Bl/6 Rag γ

P-e

IF2α

C57Bl/6 Stat1-/-

P-e

IF2α

Dnam-1+/+ Dnam-1-/-

P-e

IF2α

EResponder Non-responder

P-e

IF2α

CD

8+FO

XP

3+

100 μm

10 μm

C57Bl/6 Rag γ

P-e

IF2α

D

Figure S15

Table S1. Changes in chromosome number upon in vivo selection of hyperploid cells

DNA content(100% parental)

Chromosomalgain (%)

Chromosomal loss (%)

Tumours grown on Rag �mice

1-1 94 20 351-2 93 25 401-3 86 20 401-4 92 25 401-5 113 35 252-1 108 25 402-2 113 50 152-3 114 45 202-4 112 45 252-5 109 35 35Mean 103 ± 14 33 ± 4 32 ± 3

Tumours grown on BALB/c mice

1-1 109 30 201-2 71 15 351-3 85 25 151-4 59 20 401-5 77 20 302-1 29 5 252-2 60 20 402-3 49 20 402-4 29 0 35Mean 63 ± 29## 17 ± 3## 31 ± 3

##, p<0.01, as compared to tumours grown on Rag �mice.

Table S2. Detailed changes in chromosome number upon in vivo selection ofhyperploid tumors

Gain or loss of chromosomesComparison 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X

In vivo Rag ��

1-1 - + - - - - - + + - +1-2 - + - - - + - - - + - + +1-3 - + + - - - - - + + - -1-4 - - + + - - - - - -1-5 + - + + - - + + + - + -2-1 - - + + - - + + - - - + -2-2 + + + - + + + + + - + + -2-3 + - + - + - + + + + + + -2-4 + - + - + - + + + + + - + -2-5 + + - + - - + + + + - - - -

In vivo BALB/c

1-1 - - - - + + - - - +1-2 + + + + - - - +1-3 - + - - + - - - - + + -1-4 - - + + - - + - - +1-5 - + - + - - - - + -2-1 - - - + - -2-2 - + + - + + - - - - - -2-3 - - + - + + + - - - - -2-4 - - - - - - -

Table S3. Patient characteristics*

Responders (n=18) Non-responders (n=42) p**Age, yearsMean 47 48 0.75SD 7 11TNM stageT1 0% 9% 0.16T2 65% 41%T3 14% 29%T4 21% 21%N0 14% 35% 0.33N1 79% 59%N2 7% 6%SBR grade1 7% 6% 0.972 43% 47%3 50% 47%HRNegative 57% 47% 0.77Positive 43% 53%

* All patients (n=60) were diagnosed with invasive ductal breast carcinoma; HR, hormone receptor;

SBR, Scarf-Bloom-Richardson grade; TNM, Tumor Node Metastasis classification.

** The p value for age was calculated with the Student’s t test. p values for all other groups were

determined with the 2 test.

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