Cancer Cell, Volume 24

Supplemental Information

A Senescence-Inflammatory Switch from Cancer-Inhibitory to Cancer-Promoting Mechanism Ariel Pribluda, Ela Elyada, Zoltan Wiener, Haya Hamza, Robert E. Goldstein, Moshe Biton, Ido Burstain, Yael Morgenstern, Guy Brachya, Hana Billauer, Sharon Biton, Irit Snir-Alkalay, Domagoj Vucic, Katharina Schlereth, Marco Mernberger, Thorsten Stiewe, Moshe Oren, Kari Alitalo, Eli Pikarsky, and Yinon Ben-Neriah

Inventory of Supplemental Information 1. Supplemental Data Contain Supplemental Figures S1-S5 and legends: 

Figure S1 (relates to Figure 1)

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Figure S2 (relates to Figure 2)

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Figure S3 (relates to Figure 4)



Figure S4 (relates to Figure 6)



Figure S5 (relates to Figure 7)

2. Supplemental Experimental Procedures 3. Supplemental References

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Supplemental Data

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Supplemental Experimental Procedures Mouse breeding and genotyping CKIfl/fl;Villin-Cre-ERT2 (CKIΔgut), CKIfl/fl;p53fl/fl;Villin-Cre-ERT2 (CKIΔgut;p53Δgut) and CKIfl/fl;p21-/-;Villin-Cre-ERT2 (CKIΔgut;p21-/-) mice were generated as previously described (Elyada et al., 2011). Crude DNA extract from the ear of 4 weeks old pups was used for mouse genotyping, with the following primers: 5’- TCCACAGTTAACCGTAATCGT: forward primer for wild-type and floxed CKI; 5’- AACTGCAAATGAAAGCCCTG: reverse primer for wildtype and floxed CKI; 5’-AGCAATTCACACGTATTTGG: forward primer for wild-type p21; 5’-TGACGAAGTCAAAGTTCCACC:

reverse

AAGCCTTGATTCTGATGTGGGC:

forward

primer

for

null

p21;

5’-

GCTATCAGGACATAGCGTTGGC:

reverse

primer

for

null

p21;

5’-

primer

for

Cre;

5’-

ATGTCCAATTTACTGACCGTACACC-3’:

primer

forward

for

wild-type

p21;

5’-

CGCCTGAAGATATAGAAGATAATCG-3’: reverse primer for Cre. APCmin/+ mice (Harlan) were genotyped according to the Jackson Laboratory guidelines, and sacrificed at 3-5 months of age. Mice were kept under specific pathogen-free conditions at the Hadassah Medical School of the Hebrew University. All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Hebrew University – Hadassah Medical School, and performed in accordance with this committee’s guidelines. Tamoxifen administration, Sulindac, BV6 and BOT64 treatments, BrdU labeling and tissue preparation. Tamoxifen (Sigma) was dissolved in corn oil (Sigma) to a 20 mg/ml stock solution. Mice were injected subcutaneously with 150 mg/kg of Tamoxifen at a regimen of 6 injections every other day. Sulindac (Sigma) was administered in the drinking water at 0.6 gr/L, one week before the first Tamoxifen injection, and throughout the 2 weeks of KO induction, for a total period of 3 weeks. BV6 (Genentech) was dissolved in 15% hydroxyl-propyl--cyclodextrin/20 mM Succinic acid at pH 5.5, and was injected intraperitoneally at 10 mg/kg, every other day, from day 2 of

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KO induction. BOT64 (Santa Cruz) was dissolved in Propylene glycol/Water/Ethanol solution at 5:4:1 ratio, and was injected intraperitoneally at 30 mg/kg, every day, from day 2 of KO induction. 1-2 days after the last Tamoxifen injection, mice were injected intraperitoneally with 10 l/g of 5-bromo-2-deoxyuridine (BrdU; GE Healthcare) and sacrificed 2 hours later, on day 10-12 of knockout induction. Jejunum, ileum and the entire large intestine were flushed with icecold PBS, cut open longitudinally, and subjected to fixation in 4% formaldehyde and paraffin embedding (FFPE). Small pieces of jejunum were embedded in Tissue-Tek OCT Compound (Sakura) and frozen at –80°C, for Senescence-Associated -galactosidase (SA--gal) assay. Intestinal epithelial cells (IECs) were isolated from the middle part of the small intestine and separated into single cells in HBSS containing 10 mM HEPES, 5 mM EDTA and 0.5 mM DTT, at 37oC for 30 minutes, as previously described (Elyada et al., 2011; Greten et al., 2004). APCmin/+ mouse whole intestines were fixed in 4% formaldehyde and paraffin embedded (FFPE).

Histology, immunohistochemistry, immunofluorescence, and senescence-associated βgalactosidase analysis of intestine tissue. 5 m sections were cut from formalin-fixed parafine embedded (FFPE) blocks for hematoxylin/eosin (H&E) staining, immunohistochemistry (IHC) and immunofluorescence (IF). For IHC, sections were incubated with the following antibodies in CAS-Block (Invitrogen) or 3% BSA in TBST: CKI (C-19; 1:500; Santa Cruz), Ki67 (SP6; 1:250; Neomarkers), PanCytokeratin (1:200; DAKO), CD3 (1:100; Serotec), F4/80 (1:200; Serotec), Lamp2a (1:600; abcam), p19ARF (1:200; abcam), PTGES (1:100; abcam), Pla2g2a (1:500; Santa Cruz), BrdU (Ab3; 1:200; Neomarkers), p21 (F-5; 1:50; Santa Cruz), Prox1 (1:200; R&D), IFITM2/3-Fragilis (1:400; Abcam), p53 (CM5; 1:200; Novocastra), H2AX (1:6,000; Millipore) and Cleaved Caspase-3 (1:100; Cell Signaling). Secondary antibodies were HRP-polymer anti-mouse (mouseon-mouse; Nichirei), anti-Rat (Nichirei), anti-rabbit and anti-goat (Vector). Diaminobenzidine (DAB) chromogen (LabVision) was used for detection. For Immunofluorescence (IF), sections were incubated with a mixture of Pla2g2a and PTGES antibodies at the indicated dilutions, and 11

detected by Alexa fluor-647 donkey anti-goat and Alexa fluor-488 donkey anti-rabbit antibodies (1:1,000, Molecular Probes). Hoechst (1 g/ml; Molecular Probes) was used as nuclear counterstain. For senescence-associated -galactosidase (SA--gal) staining, 10m sections were cut from OCT-embedded frozen tissue and allowed to adhere to coated slides at room temperature for 1 min prior to fixation for 15 min in PBS/0.5% glutaraldehyde (Sigma). Sections were rinsed with PBS pH 5.5 containing 1 mM MgCl2 and incubated at 37°C overnight in prewarmed and filtered X-gal solution: 0.1% X-gal (Ornat) dissolved in PBS pH 6 containing 1 mM MgCl2, 5 mM potassium ferrocyanide and 5 mM potassium ferricyanide. Sections were rinsed with PBS, post-fixed in 95% ethanol, rehydrated, counterstained with nuclear fast red, dehydrated and mounted (Dimri et al., 1995) , or subsequently subjected to IHC detection of Ki67, Prox1 or BrdU (BU1/75; 1:600; Serotec). For APCmin/+ mouse tumor analysis, 50 adenomas from 10 mice were scored for heterogeneous CKI expression and coincident reduction in Ki67. SA--gal was examined in Ki67-low regions which were CKI-low vs. the ones that were CKI-high. Intestinal crypt cultures Intestinal crypts from WT, Apcfl/fl;Villin-Cre-ERT (APC KO), CKI KO and CKI;p53 DKO mice were isolated on the basis of the previously published method (Sato et al., 2009), with several changes: small intestines were flashed with cold PBS, cut into 5 cm pieces and opened longitudinally, washed with 70% ethanol and agitated at 2 mM EDTA in PBS for 30 minutes at 4°C. Crypts were collected as described and embedded onto Matrigel (BD Biosciences) at 50 µl/well in 24-well plates. Culture

medium was DMEM/F12 medium (Gibco), containing

Glutasmax (Gibco) and Penicillin/Streptomycin. Medium was supplemented with B27 (Gibco, 1:50), 100 ng/ml murine Noggin (Peprotech), 20 ng/ml murine EGF (Peprotech), 10 ng/ml human basic-FGF (Peprotech) and 500 ng/ml human R-Spondin1 (Peprotech). The crypts were split 1:3-1:4 and embedded onto new Matrigel every 5-7 days. Induction of knockout in the crypt cultures was carried out by incubation with 300 nM 4-hydroxy-tamoxifen (4OHT; Sigma) for 48 hours. Selection for organoids with active Wnt/-catenin pathway was done by splitting the 4OHT-induced organoids and culturing them without growth factors in the medium. Organoids were treated with: TNF (R&D, 100 ng/ml), neutralizing goat anti-mouse TNF (R&D, 2.5 g/ml), Sulindac (Sigma, 100 M or 500 M in DMSO) and BOT64 (Santa Cruz, 2.5 M in DMSO). 12

The ratio of dead or non-budding organoids was calculated on day 4. For Vital staining, Organoids were incubated with Trypan Blue for 2-5 min, washed 3 times with PBS, left in 37oC for another 15 min with PBS, washed again and visualized. For whole-mount IF staining, organoids were cultured on round cover slips in a 24-well plates. Incubation with 10 uM bromodeoxyuridine (BrdU) was performed for 16 hours in the culture medium. Fixation was performed with 4% PFA for 30 minutes. For BrdU staining, fixed organoids were treated with 2 M HCl for 25 minutes, then neutralized with 0.1 M sodium borate (pH=8.5) for 5 minutes. Organoids were permeabilized in blocking solution (1% BSA, 5% normal goat serum, 0.3% Triton X-100 in PBS) and incubated with the desired primary antibody overnight at 4°C. Primary antibodies that were used for organoid IF are: p19ARF (1:400, R&D), Lamp2a (1:100; abcam), BrdU (Ab3; 1:200; Neomarkers), p21 (F-5; 1:50; Santa Cruz) and Prox1 (1:200; R&D). Secondary antibody was applied for overnight at 25°C. DAPI or Hoechst were used as nuclei counterstain. The organoids were mounted in mounting medium and imaged with a Zeiss LSM 5 Duo confocal microscope. FACS, Western blotting and RNA analysis Immune cells (CD45.2-positive population) were sorted from intestinal epithelial cells (IECs) preparation using anti-CD45.2 antibody (1:200; BD Pharmingen). For Western blotting, wholecell protein extracts were isolated from IECs or intestinal crypt organoids. Blots were incubated with the following antibodies in 3%BSA/TBST: CKI (C-19; 1:1,000; Santa Cruz), p53 (CM5; 1:1,000; Novocastra), GSK3 (1:2,500; BD Biosciences), PP2A (1:1000; Rabbit serum provided by D. Virshup), Cyclin D1 (SP4; 1:500; Lab Vision), Cyclin D2 (1:1,000; Santa Cruz), p21 (F-5; 1:200; Santa Cruz), Hsp90 (1:5,000; Calbiochem) and Prox1 (1:200; R&D). Secondary antibodies were HRP-linked goat anti-mouse, goat anti-rabbit and rabbit anti-goat (1:10,000; Jackson). Blots were developed using ECL (GE Healthcare). Total RNA was extracted from IECs using TRI-reagent (Sigma) and phenol/chloroform methods, and from crypt cultures using NucleoSpin RNA II Kit (Macherey-Nagel). RNA (0.5-2 g) was subjected to reverse transcription using M-MLV-RT (Invitrogen), and mRNA expression levels were measured by qRT-PCR using SYBR-Green (Invitrogen) in a 7900HT Fast Real-Time PCR system (ABI). Sequences of RT-PCR primers are listed in Table S1. Relative quantities of gene transcripts were analyzed in qBase 2.2 software and normalized to UBC, HPRT or GAPDH transcripts. 13

Real-Time PCR primers

Primer

Forward (5’  3’)

Reverse (5’  3’)

Cyclin D1

TTGACTGCCGAGAAGTTGTG

CCACTTGAGCTTGTTCACCA

Cyclin D2

CACCGACAACTCTGTGAAGC

TGCTCAATGAAGTCGTGAGG

c-Myc

TGAGCCCCTAGTGCTGCAT

AGCCCGACTCCGACCTCTT

Cd44

CAGTATCTCCCGGACTGAGG

GCCAACTTCATTTGGTCCAT

Sox9

GGAGCTCAGCAAGACTCTGG

TGTAATCGGGGTGGTCTTTCT

Bax

ATGCGTCCACCAAGAAGCTGA

AGCAATCATCCTCTGCAGCTCC

Cyclin G1

GCTGGCGCTATCTATCCTTG

GGTCAAATCTCGGCCACTTA

p21

TCCACAGCGATATCCAGACA

AGACAACGGCACACTTTGCT

Tnf

ACCACGCTCTTCTGTCTACTGAA

TCCCTCTCATCAGTTCTATGGC

Tlr1

GGACCTACCCTTGCAAACAA

TATCAGGACCCTCAGCTTGG

Tlr2

GAGCATCCGAATTGCATCA

ACAGCGTTTGCTGAAGAGGA

Il1rn

TTGTGCCAAGTCTGGAGATG

TTCTCAGAGCGGATGAAGGT

Troy

CGCTGCCATTCTCTTCCTAC

TCGATCCTTGAATTCCTGCT

p19Arf

GTCACACGACTGGGCGATT

GACTCCATGCTGCTCCAGAT

Opg

ATGAACAAGTGGCTGTGCTG

TCACACAGGAGCTGATGACC

Plastin3

TGGAGAGGGTCAGAAAGCAAA

AATCCACAACCGCCAAACTG

Ifitm1

ATCTCCACGCCTGACCATGT

CACCCACCATCTTCCTGTCC

Ifitm3

CTGCTGCCTGGGCTTCATAG

GGATGCTGAGGACCAAGGTG

Prox1

ATACCGAGCCCTCAACATGC

CGTAACGTGATCTGCGCAAC

Plat

AGTTCCTGCTGGGTGCTGTC

CGGGGACCACCCTGTATGTT

Oas2

CCCTGTGAAGGAAGTGGCTA

CTGTTGGAAGCAGTCCATGA

Tnfrsf1b

GTCTTCGAACTGCAGCTGTG

TACCCAGGTTCCGGTTTGTA

Lpo

TGACCTTGCTCCAGACTGC

TTGACCCAGACCTTGACCTC

Faim2

CTCGAGAGAAGACATCATGACC

TTCTCTCCATTTGCCTGGTG

Sox4

AATTGCACCAACTCCTCAGC

TCGATTGCAGTTCACGAGAG

Lass3

GAGAACAAAGCTGCCTGGAA

AGCCAGTATCTCTCCGACAA

Igfbp4

GGAGCTGTCGGAAATCGAAG

TTGAAGCTGTTGTTGGGATG

Tnfrsf8

GAGACTCGGGAAGCCAAGAT

GGTGGTCTTGAGTGGTCGAT

Pla2g2a

TTAAGACAGGAAAGAGAGCTGAGC

GTACCACATCCACTTTTCTCCAG

Slc7a11

TCTGGTCTGCCTGTGGAGTA

CAAAGGACCAAAGACCTCCA

Sox17

TGAAATATGGCCCACTCACA

CTGTCTTCCCTGTCTTGGTTG

Cd40

GCAGTGTGTTACGTGCAGTG

CTGTGCAGTGGCTTGTCAGT

Ptges

AGCACACTGCTGGTCATCAA

TCCACATCTGGGTCACTCCT

Ly6a

TACCTGCCCCTACCCTGATG

AGGAGGGCAGATGGGTAAGC

Axin2

CTCCCCACCTTGAATGAAGA

ACTGGGTCGCTTCTCTTGAA

Gapdh

ACAACTTTGGCATTGTGGAA

GATGCAGGGATGATGTTCTG

14

Ubc

CAGCCGTATATCTTCCCAGACT

CTCAGAGGGATGCCAGTAATCTA

Hprt

GTTAAGCAGTACAGCCCCAAA

AGGGCATATCCAACAACAAACTT

RNA-Seq For RNAseq, total RNA from IECs of three mice per genotype was pooled, processed with the mRNA Sequencing Kit (Illumina) and subjected in duplicates to multiplex sequencing on the Illumina HiSeq2000 platform, according to the manufacturer's protocol. Unique 50 bp single reads were aligned to the mouse genome Ensembl revision 70 using TopHat (Trapnell et al., 2009). Read counts were normalized and gene expression in SKO and DKO was calculated as log2-fold change compared to CKI heterozygotes. The RNAseq full data can be found at: http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1683/

Supplemental References Dimri, G.P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens, M., Rubelj, I., Pereira-Smith, O., et al. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92, 9363-9367. Subramanian, A., Tamayo, P., Mootha, V.K., Mukherjee, S., Ebert, B.L., Gillette, M.A., Paulovich, A., Pomeroy, S.L., Golub, T.R., Lander, E.S., et al. (2005). Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102, 15545-15550. Trapnell, C., Pachter, L., and Salzberg, S.L. (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105-1111.

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