LETTERdoi:10.1038/nature13638

Tumour-infiltrating Gr-11 myeloid cells antagonizesenescence in cancerDiletta Di Mitri1*, Alberto Toso1*, Jing Jing Chen1,2, Manuela Sarti1, Sandra Pinton1, Tanja Rezzonico Jost3, Rocco D’Antuono3,Erica Montani3, Ramon Garcia-Escudero1,4, Ilaria Guccini1, Sabela Da Silva-Alvarez5, Manuel Collado5, Mario Eisenberger6,Zhe Zhang7, Carlo Catapano1, Fabio Grassi3,8 & Andrea Alimonti1,2

Aberrant activation of oncogenes or loss of tumour suppressor genesopposes malignant transformation by triggering a stable arrest incell growth, which is termed cellular senescence1–3. This process isfinely tuned by both cell-autonomous and non-cell-autonomousmechanisms that regulate the entry of tumour cells to senescence4–6.Whether tumour-infiltrating immune cells can oppose senescenceis unknown. Here we show that at the onset of senescence, PTEN nullprostate tumours in mice2,7 are massively infiltrated by a populationof CD11b1Gr-11 myeloid cells that protect a fraction of proliferat-ing tumour cells from senescence, thus sustaining tumour growth.Mechanistically, we found that Gr-11 cells antagonize senescence ina paracrine manner by interfering with the senescence-associatedsecretory phenotype of the tumour through the secretion of inter-leukin-1 receptor antagonist (IL-1RA). Strikingly, Pten-loss-inducedcellular senescence was enhanced in vivo when Il1ra knockout mye-loid cells were adoptively transferred to PTEN null mice. Therapeu-tically, docetaxel-induced senescence and efficacy were higher in PTENnull tumours when the percentage of tumour-infiltrating CD11b1Gr-11 myeloid cells was reduced using an antagonist of CXC chemokinereceptor 2 (CXCR2)8. Taken together, our findings identify a novelnon-cell-autonomous network, established by innate immunity, thatcontrols senescence evasion and chemoresistance. Targeting this net-work provides novel opportunities for cancer therapy.

Cellular senescence is a stable state of cell growth arrest that opposestumour initiation and progression in a variety of in vivo tumour models1,2.Recent studies have revealed an unexpected role for both adaptive andinnate immunity in the regulation of cellular senescence. Immune cellscan either clear senescent cells from tumours or induce senescence in can-cer cells by secreting pro-inflammatory cytokines9–11. However, whethertumour-infiltrating immune cells can also oppose senescence in vivo isnot known. We have previously shown that complete inactivation of thetumour suppressor gene Pten in the mouse prostate epithelium inducesthe formation of benign tumours characterized by a strong senescenceresponse that opposes tumour progression2,7. However, these tumoursgrow over time and progress to become more aggressive and invasivetumours2,12. Indeed, at the onset of senescence (7–8 weeks), PTEN nulltumours (hereafter referred to as Ptenpc2/2 tumours) are characterizedby the concomitant presence of both senescent and proliferative cellularcompartments, as shown by the expression of p16INK4A (also known asCDKN2A), pHP1c (also known as CBX3) and senescence-associatedb-galactosidase (SA-b-gal) and by Ki-67 positivity, respectively (Fig. 1aand Extended Data Fig. 1a–c). This finding suggests that from the earlystages of tumorigenesis, a fraction of proliferating tumour cells evadessenescence. Given the interplay between senescent tumour cells andimmune cells, we speculated that Ptenpc2/2 tumours may evade senescence

in a non-cell-autonomous manner and that the tumour microenviron-ment could be the source of factors that hinder the senescence response.

To address this hypothesis, we first characterized the immune micro-environment of Ptenpc2/2 tumours at the onset of senescence and founda strong infiltration of CD451CD11b1Gr-11 myeloid cells (hereafterreferred to as Gr-11 cells) (Fig. 1b and Extended Data Fig. 1d–f). More-over, the prostate lobes with the highest percentage of Ki-67 stainingwere the most infiltrated by Gr-11 cells (Extended Data Fig. 1g). Tostudy the localization of tumour-infiltrating Gr-11 cells in the prostate,we adoptively transferred bone marrow precursors from mice transgenicfor green fluorescent protein (GFP) under control of the human ubiquitinC (UBC) promoter13 into lethally irradiated Ptenpc2/2 mice (ExtendedData Fig. 1h). Immunofluorescence showed that GFP1 cells localized tothe stroma and the prostate glands (Fig. 1c and Extended Data Fig. 1i, j).Moreover, GFP1 cells were localized in close proximity to proliferating(Ki-671) tumour cells (Fig. 1c and Extended Data Fig. 1j). Immune cellssecrete a variety of cytokines that regulate senescence11. Interestingly,the majority of GFP1 cells were spatially distributed within 100 mm ofKi-671 epithelial cells (Fig. 1c), suggesting that GFP1 epithelial cells mayinterfere with senescence in a paracrine manner14,15. Notably, ,70% ofthe tumour-infiltrating GFP1 cells expressed the myeloid differentia-tion antigen Gr-1 (Fig. 1d and Extended Data Fig. 1k). We also confirmedthese results in tumour sections from non-irradiated Ptenpc2/2 mice16

(Extended Data Fig. 1l). To assess whether factors secreted by Gr-11 cellscan oppose Pten-loss-induced cellular senescence, we cultured Pten2/2

mouse embryonic fibroblasts (MEFs), which undergo senescence in vitro2,7,in the presence of conditioned medium obtained from Gr-11 myeloidcells17 (Extended Data Fig. 1m, n). Surprisingly, Pten-loss-induced cel-lular senescence was impaired in Pten2/2 MEFs cultured in the pres-ence of conditioned medium from Gr-11 cells (Fig. 1e and Extended DataFig. 1o). These data demonstrate that Gr-11 cells oppose senescence ina paracrine manner.

To identify secreted factors that mediate the anti-senescence functionof Gr-11 cells, we compared the cytokine profile of Ptenpc2/2 tumoursbefore and after depletion of immune cells18 and found that IL-1RA wasthe cytokine present at the most reduced level after immunodepletion(Extended Data Fig. 2a and Supplementary Table 1). Gene expressionanalysis, quantitative PCR with reverse transcription (qRT–PCR) andimmunofluorescence confirmed that Gr-11 cells were the major sourceof IL-1RA in Ptenpc2/2 tumours relative to epithelia (Fig. 2a, b andExtended Data Fig. 2b). It should also be noted that CD11b1Gr-12F4/801 cells released IL-1RA in the tumour microenvironment (ExtendedData Fig. 2c). IL-1RA is an antagonist of IL-1R and has been reported toimpair oncogene-induced senescence in vitro by blocking IL-1a-mediatedsignalling5,6. Notably, in Ptenpc2/2 tumours, the prostate lobes with the

*These authors contributed equally to this work.

1Institute of Oncology Research (IOR), Oncology Institute of Southern Switzerland, Bellinzona CH6500, Switzerland. 2Faculty of Biology and Medicine, University of Lausanne UNIL, Lausanne CH1011,Switzerland. 3Institute for Research in Biomedicine (IRB), Bellinzona CH6500, Switzerland. 4Molecular Oncology Unit, CIEMAT, 28040 Madrid, Spain. 5Laboratory of Stem Cells in Cancer and Aging,(stemCHUS) Health Research Institute of Santiago de Compostela (IDIS), Clinical University Hospital (CHUS), E15706 Santiago de Compostela, Spain. 6Department of Oncology, Sidney KimmelComprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland 21231-1000, USA. 7Divisions of BioStatistics, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University,Baltimore, Maryland 21231-1000, USA. 8Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan I-20100, Italy.

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highest percentage of Gr-11 myeloid cells (Extended Data Fig. 1g) dis-played the highest levels of IL-1RA and the lowest levels of p16INK4A

(Extended Data Fig. 3a). To elucidate the role of IL-1a-mediated signal-ling in Pten-loss-induced cellular senescence, we cultured Pten2/2 MEFsin the presence of IL-1RA. While IL-1awas strongly expressed in senes-cent Pten2/2 cells, unlike in Pten1/1 cells, the expression of IL-1RA wasslightly higher than in Pten1/1 cells (Fig. 2c). Remarkably, treatmentwith IL-1RA decreased both SA-b-gal staining and levels of the tumoursuppressor protein p53 in Pten2/2 cells (Fig. 2d, e). In-vitro-polarizedGr-11 myeloid cells expressed high levels of Il1ra (Extended Data Fig. 3b).When Pten2/2 MEFs were cultured in the presence of conditioned med-ium from Gr-11 myeloid cells derived from Il1ra knockout (Il1ra2/2)mice19,20, Pten-loss-induced cellular senescence was not impaired (Fig. 2f, g).Similar results were obtained in MEFs transfected with Ha-rasV12 (H-ras), suggesting that myeloid cells also oppose oncogene-induced senes-cence (Extended Data Fig. 3c, d). Moreover, conditioned medium fromGr-11 cells pre-treated with a JAK2 inhibitor failed to block Pten-loss-induced cellular senescence21 (Extended Data Fig. 3e, f). Importantly,IL-1RA also blocked docetaxel-induced senescence22 in human prostatecancer cells (Extended Data Fig. 4a, b).

We next validated our findings in a mouse model of oncogene-inducedsenescence. Ki-ras1/G12V mice develop both lung adenomas and ade-nocarcinomas that display stage-dependent expression of senescencemarkers23. Interestingly, lung adenocarcinomas were characterized bythe infiltration of Gr-11 myeloid cells, IL-1RA expression and Ki-67positivity and the absence of senescence markers (Extended Data Fig. 5a).By contrast, senescent lung adenomas were poorly infiltrated by mye-loid cells (Extended Data Fig. 5a–c). These data suggest that myeloid cellsmay oppose senescence in different types of tumour, irrespective of thegenetic background. To validate our findings in vivo, we adoptively trans-ferred bone marrow precursors from Il1ra1/1 or Il1ra2/2 mice intolethally irradiated Ptenpc2/2 mice (yielding Ptenpc2/2Il1ra1/1 mice andPtenpc2/2Il1ra2/2 mice) (Fig. 3a and Extended Data Fig. 6a). Notably,Ptenpc2/2Il1ra1/1 and Ptenpc2/2Il1ra2/2 tumours were infiltrated equallyby Gr-11 cells (Fig. 3b). Strikingly, histopathological analysis revealedthat Ptenpc2/2Il1ra2/2 tumours displayed an almost complete normal-ization of glands affected by prostatic intraepithelial neoplasia (Fig. 3c, d),which was associated with decreased cell proliferation, increased senes-cence and absence of apoptosis (Fig. 3c, e and Extended Data Fig. 6b–h),in contrast to Ptenpc2/2Il1ra1/1 tumours. Finally, the enhanced senes-cence response in Ptenpc2/2Il1ra2/2 tumours was associated with theactivation of IL-1a-mediated signalling5 (Fig. 3f). Myeloid cells were adop-tively transferred to Ptenpc2/2 mice in the absence of T cells (ExtendedData Fig. 7a, b), suggesting that Gr-11 myeloid cells oppose senescencethrough a novel pro-tumorigenic mechanism that does not involve thiscell population8,24. These results demonstrate that Gr-11 myeloid cellsantagonize senescence in vivo in a paracrine manner by interfering with

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Figure 2 | Gr-11 myeloid cells oppose Pten-loss-induced cellular senescenceby interfering with IL-1a signalling in vitro. a, mRNA levels of secretedfactors (n 5 3 per group). b, Representative confocal images of GFP1 myeloidcells co-expressing Gr-1 (red) and IL-1RA (grey) (nuclei, blue (DAPI)). Scalebar, 5 mm. c, Il1a and Il1ra mRNA levels in Pten1/1 and Pten2/2 MEFs (n 5 3).d, Percentage of SA-b-gal1 cells in Pten1/1 and Pten2/2 MEFs treated withrecombinant IL-1RA (n 5 4). e, Western blot for p53 in MEFs treated withrecombinant IL-1RA. Numbers indicate fold changes in protein level.f, Percentage of SA-b-gal1 cells in Pten1/1 and Pten2/2 MEFs cultured in thepresence of conditioned medium from Il1ra1/1Gr-11 or Il1ra2/2Gr-11 cells(n 5 5). g, Western blot for p53. Numbers indicate fold changes in protein level.a, c, d, f, Error bars, mean 6 s.e.m. P values were derived from an unpaired,two-tailed Student’s t-test (*P , 0.05; **P , 0.01).

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Figure 1 | Gr-11 myeloid cells oppose Pten-loss-induced cellularsenescence. a, Haematoxylin and eosin (H&E), Ki-67 and p16INK4A

immunohistochemical staining (Ki-67 and p16INK4A, blue; nuclei, brown) (left)and quantification (right) in tumours from 8-week-old Ptenpc1/1 andPtenpc2/2 mice (n 5 15 mice, 1 tumour assessed per mouse, 3 sections assessedper tumour, $5 fields assessed per section). Original magnification, 3400.b, Flow cytometry plots of CD11b1Gr-11 immune cells in tumours from8-week-old Ptenpc1/1 and Ptenpc2/2 mice (n 5 6), gating on CD451 cells.c, Confocal images (left) and quantification (right) of the localization anddistance between tumour-infiltrating myeloid cells (GFP1, green) andproliferating epithelial cells (cytokeratin 18 (CK18), grey; Ki-67, red) inPtenpc2/2UBC-GFP prostate lesions (nuclei, blue (DAPI)) (n 5 4 mice, 1tumour per mouse, 5 fields acquired, 320 cells measured). The arrow headspoint to positions where UBC-GFP1 cells colocalize in close proximity to Ki-671 cells. Scale bar, 10 mm. d, Representative confocal images of UBC-GFP1Gr-11 cells in Ptenpc2/2UBC-GFP prostate lesions (nuclei, blue (DAPI)).Scale bar, 5 mm. e, Quantification of SA-b-gal staining in Pten1/1 and Pten2/2

cells (n 5 5). CM, conditioned medium; UT, untreated. f, Cell proliferationof Pten1/1 and Pten2/2 cells (fold change compared with UT Ptenpc1/1)(n 5 5). a, c, e, f, Error bars, mean 6 s.e.m. P values were derived from anunpaired, two-tailed Student’s t-test (*P , 0.05; ***P , 0.001).

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IL-1a-mediated signalling. Moreover, Ptenpc2/2 tumours from micetreated with IL-1a showed a significant reduction in the percentage ofglands affected by prostatic intraepithelial neoplasia and the numberof Ki-671 cells and a strong increase in p16INK4A expression (ExtendedData Fig. 8a–e). This finding suggests that IL-1amainly plays a tumoursuppressive role in Pten-loss-induced cellular senescence.

Different types of chemotherapy drug are known to induce senescencein tumours25. Therefore, we reasoned that chemotherapy-induced senes-cence could also be weakened by tumour-infiltrating Gr-11 myeloidcells. Notably, the chemokines CXCL1 and CXCL2, which act throughthe chemokine receptor CXCR2 to recruit Gr-11 myeloid cells8, werestrongly upregulated in Ptenpc2/2 tumours (Supplementary Table 1 andExtended Data Fig. 8f). We next combined docetaxel treatment, whichdrives senescence in tumours22, with an antagonist of CXCR2 to blockthe recruitment of Gr-11 myeloid cells to Ptenpc2/2 tumours (ExtendedData Fig. 8g–j). Our pre-clinical study showed that treatment with theCXCR2 antagonist synergized with docetaxel. Indeed, in mice treatedwith the CXCR2 antagonist and docetaxel, we observed a strong anti-tumour response and a concomitant reduction in the IL-1RA levels(Fig. 4a, b and Extended Data Fig. 9a). These changes were associatedwith an enhanced senescence response, reduced proliferation and theabsence of apoptosis (Fig. 4c–e and Extended Data Fig. 9b–d). Notably,treating Pten2/2 MEFs with the CXCR2 antagonist did not affect senes-cence (Extended Data Fig. 9e, f). Next, we assessed the levels of IL-1RA

in primary tumours from patients with high-risk, localized prostate cancerwho received docetaxel after prostatectomy in a prospective multicen-tre trial26. Interestingly, patients with high levels of intratumoral IL-1RA did not respond to docetaxel and had a short disease-free survival(15 6 10 months, mean6 s.e.m.; P 5 0.04) compared with patients withnormal IL-1RA levels (216 13 months) (Extended Data Fig. 9g). Finally,we looked at the correlation between tumour-infiltrating CD331 mye-loid cells and p16INK4A1 senescent cells in a human tissue microarrayof prostate cancer (n 5 92 cases) and found that the majority of tumoursamples infiltrated by CD331 myeloid cells stained negative for p16INK4A

(Fig. 4f and Extended Data Fig. 10a). This result was also confirmed in apanel of single human prostate cancer sections (n 5 18), including areasof prostatic intraepithelial neoplasia and prostate cancer (Extended DataFig. 10b). Moreover, we found that the majority of the prostatic intrae-pithelial neoplasia areas analysed had high p16INK4A staining and lowCD33 and Ki-67 staining. Conversely, 90% of the prostate cancer areasstained negative for p16INK4A and positive for CD33 and Ki-67 (Fig. 4gand Extended Data Fig. 10c). These findings suggest that myeloid cellsmay promote tumour progression by opposing senescence in cancer inhumans, in addition to mice. Bioinformatic analysis of data from the Pan-Cancer analysis project revealed that patients with low levels of PTEN

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Figure 3 | Adoptively transferred Il1ra2/2 bone marrow precursorsenhanced Pten-loss-induced cellular senescence in vivo. a, Experimentalset-up. NK, natural killer. b, Flow cytometry plots showing equal infiltration ofGr-11 cells in Ptenpc2/2Il1ra1/1 and Ptenpc2/2Il1ra2/2 mice. c, H&E, Ki-67and p16INK4A immunohistochemical staining of sections from 12-week-oldPtenpc2/2Il1ra1/1 and Ptenpc2/2Il1ra2/2 mice. Ki-67 and p16INK4A, brown;nuclei, blue. Original magnification, 3400. d, Quantification of prostaticintraepithelial neoplasia (PIN)-affected glands in Ptenpc2/2Il1ra1/1 andPtenpc2/2Il1ra2/2 mice. e, Confocal images of staining for CK18 (grey) andpHP1c (green) in prostate tumours from the indicated genotypes (nuclei, blue(DAPI)). Percentage of cells that stained positive for pHP1c, 15 6 7% (bottomleft) and 37 6 13% (bottom right). Original magnification, 3400. f, IL-1asignalling. Fold change in the expression of IL-1 target genes in tumours of theindicated genotypes. d, f, n 5 4; 1 tumour per mouse; 3 sections per mouse;$5 fields per section. Error bars, mean 6 s.e.m. P values were derived from anunpaired, two-tailed Student’s t-test (*P , 0.05; **P , 0.01).

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tumours: relevance for human cancer. a, b, Tumour volume andquantification of PIN-affected glands in Ptenpc2/2 mice treated withdocetaxel or a CXCR2 antagonist (CXCR2a). c–e, Ki-67 and p16INK4A

immunohistochemical staining (Ki-67 and p16INK4A, brown; nuclei, blue)(c) and Ki-67 (d) and p16INK4A (e) quantification in Ptenpc2/2 mice treatedwith docetaxel, CXCR2a or both. Scale bar, 10 mm. f, CD33 and p16INK4A

immunohistochemical staining in consecutive tissue microarray (n 5 92)sections of human prostate cancer from two donors. CD33 and p16INK4A,brown; nuclei, blue. Main images, magnification 3 400; insets, magnification3 100. g, p16INK4A and CD33 immunohistochemical staining in sections ofPIN and prostate cancer (PCA) from the same donor. CD33 and p16INK4A,brown; nuclei, blue. Main images, magnification 3 400; insets, Ki-67 positivecells in the same region at the same magnification. a, b, d, e, n 5 5 controlgroup; n 5 7 treated groups; 1 tumour per mouse; 3 sections per mouse;$5 fields per section. Error bars, mean 6 s.e.m. P values were derived from anunpaired, two-tailed Student’s t-test (*P , 0.05; **P , 0.01; ***P , 0.001).

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and high levels of IL1RA and CD33 messenger RNA had a shorter sur-vival than the other groups (Extended Data Fig. 10d).

Taken together, our data allow novel insight into the mechanisms thatregulate senescence in vivo (Extended Data Fig. 10e). Here we provide, toour knowledge, the first evidence that an immune cell subset can antag-onize senescence driven by loss of a tumour suppressor gene in vivo,demonstrating that senescence evasion by tumour cells can also occurin a non-cell-autonomous manner. This finding is of great relevancesince senescence evasion is commonly ascribed to genetic alterationsthat are unlikely to occur with high frequency in senescent arrested cellsor in cells committed to senescence10,27–29.

Our study supports a model whereby Gr-11 myeloid cells protectPten2/2 tumour cells from senescence by interfering with the balancebetween IL-1a and IL-1RA in the tumour microenvironment (ExtendedData Fig. 10f). Accordingly, treatments that block the recruitment ofGr-11 cells or decrease the levels of IL-1RA can tilt the IL-1a to IL-1RAbalance, reinforcing senescence in tumours and enhancing the efficacyof chemotherapy.

Online Content Methods, along with any additional Extended Data display itemsandSourceData, are available in the online version of the paper; references uniqueto these sections appear only in the online paper.

Received 14 October 2013; accepted 1 July 2014.Published online 24 August; corrected online 5 November 2014 (see full-text HTMLversion for details).

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18. Lukacs, R. U., Goldstein, A. S., Lawson, D. A., Cheng, D. & Witte, O. N. Isolation,cultivation and characterization of adult murine prostate stem cells. NatureProtocols 5, 702–713 (2010).

19. Horai, R. et al. Production of mice deficient in genes for interleukin (IL)-1a, IL-1b,IL-1a/b, and IL-1 receptor antagonist shows that IL-1b is crucial in turpentine-induced fever development and glucocorticoid secretion. J. Exp. Med. 187,1463–1475 (1998).

20. Sgroi, A. et al. Interleukin-1 receptor antagonist modulates the early phaseof liver regeneration after partial hepatectomy in mice. PLoS ONE 6, e25442(2011).

21. Tamassia, N.et al. Uncoveringan IL-10-dependent NF-kB recruitment to the IL-1rapromoter that is impaired in STAT3 functionally defective patients. FASEB J. 24,1365–1375 (2010).

22. Schwarze, S. R., Fu, V. X., Desotelle, J. A., Kenowski, M. L. & Jarrard, D. F. Theidentification of senescence-specific genes during the induction of senescence inprostate cancer cells. Neoplasia 7, 816–823 (2005).

23. Collado, M. et al. Tumour biology: senescence in premalignant tumours. Nature436, 642 (2005).

24. Gabrilovich,D. I.&Nagaraj, S.Myeloid-derived suppressor cells as regulatorsof theimmune system. Nature Rev. Immunol. 9, 162–174 (2009).

25. Ewald, J. A.,Desotelle, J. A., Wilding,G.& Jarrard,D. F. Therapy-inducedsenescencein cancer. J. Natl. Cancer Inst. 102, 1536–1546 (2010).

26. Antonarakis, E. S. et al. An immunohistochemical signature comprising PTEN,MYC, andKi67 predicts progression inprostate cancerpatients receiving adjuvantdocetaxel after prostatectomy. Cancer 118, 6063–6071 (2012).

27. Jacobs, J. J. et al. Senescence bypass screen identifies TBX2, which repressesCdkn2a (p19ARF) and is amplified in a subset of human breast cancers. NatureGenet. 26, 291–299 (2000).

28. Romanov, S. R. et al. Normal human mammary epithelial cells spontaneouslyescape senescence and acquire genomic changes. Nature 409, 633–637 (2001).

29. Berns, K.et al. A large-scaleRNAi screen inhuman cells identifiesnewcomponentsof the p53 pathway. Nature 428, 431–437 (2004).

Supplementary Information is available in the online version of the paper.

Acknowledgements We thank L. Buhler for providing the Il1ra knockout mice, theF. Grassi laboratory and all members of the IRB animal core facility for technicalassistanceandtheanimalwork, F.Stoffel forprovidinghuman samples,D. Jarrossay forhelping with the flow cytometry analysis and cell sorting experiments, and allmembersof the Alimonti laboratory for scientific discussions. We thank C. Pissot-Soldermann,who developed NVP-BSK805. We thankT. Radimersky for providing NVP-BSK805. Thehuman tissuemicroarray (asdescribed in ref.26)wasobtained fromtheDepartment ofDefense Prostate Cancer Research Program, Awards W81XWH-10-2-0056 andW81XWH-10-2-0046, Prostate Cancer Biorepository Network (PCBN). We thankG. Chiorino, I. Gregnanin, P. Ostano and L. Sacchetto for the gene expression analysisperformed on myeloid and tumour cells. This work was supported by Swiss nationalscience foundation (SNF) grant Ambizione (PZ00P3_136612/1), the EuropeanSociety for Medical Oncology (ESMO) translational research award to A.A., the SwissBridge Award to A.A., PEOPLE-IRG (22484), a European Research Council startinggrant (ERCsg 261342), ABREOC, the Train COFUND Marie Curie to D.D.M. andFondazione IBSA.

Author Contributions A.A. and A.T. originally developed theconcept, furtherelaboratedon it and designed the experiments together with D.D.M. D.D.M., A.T. and J.J.C.performed experiments and analysed the data. R.D., E.M., A.T. and D.D.M. establishedand carried out fluorescence microscopy. D.D.M. and T.R.J. carried out adoptivetransfer experiments.M.S. andS.P.performed immunohistochemical experiments andanalysis. I.G. performed experiments, R.G.-E. and C.C. carried out the bioinformaticsanalysis. S.D.S.-A. and M.C. provided the K-ras1/G12V tumour samples. M.E. and Z.Z.provided tumour samples for the human prostate cancer study. A.T., D.D.M., F.G. andA.A. interpreted the data and wrote the paper. D.D.M. and A.T. contributed equally tothis work.

Author Information The geneexpressiondatahavebeen deposited inGene ExpressionOmnibus under accession number GSE58413. Reprints and permissions informationis available at www.nature.com/reprints. The authors declare no competing financialinterests. Readers are welcome to comment on the online version of the paper.Correspondence and requests for materials should be addressed toA.A. (andrea.alimonti@ior.iosi.ch).

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METHODSAnimals. All mice were maintained under specific-pathogen-free conditions inthe animal facilities of the IRB Institute, and experiments were performed accord-ing to state guidelines and approved by the local ethics committee. Male Ptenpc2/2

and Ptenpc1/2 mice were generated and genotyped as previously described2,12. MaleCByJ.B6-Tg(UBC-GFP)30Scha/J13 transgenic mice that express GFP under the UBCpromoter were provided by F. Grassi and were genotyped as previously described (atIRB Bellinzona)13. Male Il1ra knockout mice (Il1ra2/2) were provided by L. Buhlerand were genotyped as previously described20.

For experiments involving animals, the sample size was chosen taking into con-sideration the means of the target values between the experimental group and thecontrol group, the standard deviation and the statistical analysis used. For ethicalreasons, the minimum number of animals necessary to achieve the scientific objec-tives was used. Animals were allocated randomly to each treatment group. Differenttreatment groups were processed identically, and animals in different treatmentgroups were exposed to the same environment.Human prostate samples. Human tissue microarray (TMA) analyses were carriedout using previously published data sets26 and commercially available TMAs. Anony-mized human tissue samples were obtained from the Cantonal Institute of Pathology(Locarno, Switzerland). Informed consent was obtained from all subjects. The studywas approved by the Canton Ticino Ethics Committee. Three-micrometre sectionswere cut from formalin-fixed paraffin-embedded (FFPE) blocks and mounted onpositively charged slides. Histological classification was carried out on slides stainedwith haematoxylin and eosin. The histological diagnosis was determined during rou-tine pathological assessment. Slides were blindly evaluated by at least two investigators.Cells. Primary MEFs were derived from littermate embryos and obtained by cross-ing PtenloxP/loxP animals as previously described7,30. Embryos were harvested at 13.5days post coitum, and individual MEFs were produced and cultured as previouslydescribed2,12. At passage 2, the cells were harvested for western blot analysis. In theculture experiments, conditioned medium were from either Il1ra1/1 or Il1ra2/2

cultures. In some experiments, bone marrow precursors were collected from thefemurs of Pten1/1 mice and polarized in vitro in the presence of granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-6 towards the Gr-11 cellphenotype. On day 4, the conditioned medium from Gr-11 myeloid cells was col-lected, filtered and transferred to Pten2/2 MEFs. Cultures were stopped 48 h later,and cells were harvested for protein extraction or stained for analysis (see also Ex-tended Data Fig. 1m for scheme). Senescence was assessed by means of an SA-b-galassay (Cell Signaling Technology). Gr-11 cells were pre-treated for 24 h with NVP-BSK805 (a JAK2 inhibitor).

For all of the in vitro experiments, the sample size was chosen taking into con-sideration the means of the target values between the experimental group and thecontrol group, the standard deviation and the statistical analysis used. Most of thein vitro experiments were performed in a non-blinded manner. Nevertheless, insome cases, the results were collected by an investigator other than the one whoperformed the experiment, to ensure blinded evaluations.In vitro differentiation of Gr-11 myeloid cells. Gr-11 cells were differentiated invitro as previously described17. Briefly, bone marrow precursors were flushed fromthe long bones of Pten1/1 mice or UBC-GFP mice with RPMI 1640 medium. Thecell pellet was resuspended (1 3 106 cells ml21) in RPMI 1640 containing 10% heat-inactivated FBS, and the cells were cultured in vitro in the presence of 10 ng ml21

GM-CSF and 40 ng ml21 IL-6. On day 4, the cells were harvested and analysed byflow cytometry and qPCR (see also Extended Data Fig. 1m for scheme).Bone marrow transplantation. Bone marrow was flushed from the long bones ofmale Il1ra1/1 or Il1ra2/2 mice under sterile conditions with RPMI 1640 or HBSSusing a 21-gauge needle. Mononuclear cells were filtered, collected and checked forviability using trypan blue. Before transplantation, the bone marrow derived fromdonor mice was depleted of CD31 T cells, NK1.11 NK cells and CD191 B cells bymagnetic bead separation. Recipient C57BL/6 Ptenpc2/2 mice were given 900 cGytotal-body irradiation (at 7–8 weeks of age), and all mice received an eye inoculumcomprising 4.03 106 bone marrow cells from either Il1ra1/1 or Il1ra2/2 mice. Bonemarrow precursors were delivered 2 h after irradiation. All mice (n 5 4 per group)survived (see also the scheme in Fig. 3a).Western blotting, immunohistochemistry and immunofluorescence. Tissueand purified epithelial lysates were prepared with RIPA buffer (13 PBS, 1% NP-40,0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (Roche)). Thefollowing antibodies were used for western blotting: rabbit polyclonal anti-p16INK4A

(M156; Santa Cruz Biotechnology), and mouse monoclonal anti-b-actin (AC-74;Sigma), anti-PAI1 (H-135; Santa Cruz Biotechnology), anti-IL-1RA (H-110; SantaCruz Biotechnology) and anti-E-cadherin (36/E-cadherin; BD Biosciences). Forimmunohistochemistry (IHC), tissues were fixed in 10% formalin and embeddedin paraffin in accordance with standard procedures. Sections were stained with anti-p53 (Accurate Chemical), anti-p16INK4A (M156; Santa Cruz Biotechnology), anti-Ki-67 (clone SP6; Lab Vision), anti-PAI1 (H-135; Santa Cruz Biotechnology) or

anti-cleaved-caspase-3 (9661, Cell Signaling Technology) antibodies. Immunofluo-rescence (IF) on paraffin-embedded sections was conducted with anti-vimentin(RV202; Abcam), anti-Ki-67 (clone SP6; Lab Vision) and anti-Ly-6G (Gr-1) anti-bodies. Confocal images were obtained with a TCS SP5 confocal microscope (Leica).Prostatic epithelial cell purification and cytokine array. Eight-week-old Ptenpc1/1

and Ptenpc2/2 mice were euthanized, and whole prostates were isolated and pro-cessed to single-cell suspensions18 for magnetic-activated cells sorting (MACS).Single cells were stained with FITC–anti-CD34 (stroma), FITC–anti-Ter119 (ery-throcytes), FITC–anti-CD31 (endothelial cells) and FITC-anti-CD45 (leukocytes)antibodies and incubated for 20 min on ice. All antibodies (BD Biosciences) wereused at 1:300; cells were then loaded onto an MS column (Miltenyi Biotec) for MACSseparation, and unstained epithelial cells were collected in the negative fraction.Purified prostatic epithelial cells were processed as indicated by the manufacturer’sinstructions in the cytokine array kit (R&D Systems). Developed films were scanned,and the obtained images were analysed using ImageJ 1.43u; background signals weresubtracted from the experimental values.Osmotic pump implantation. Micro-osmotic pumps filled with PBS or recom-binant IL-1a (3 mg kg21) were implanted in the peritoneal cavity of two groups ofage-matched Ptenpc2/2 mice, to expose the prostate tissue to a continuous and con-trolled concentration of vehicle or protein, respectively. Briefly, mice were anaes-thetized, and a midline skin incision was made in the lower abdomen. A pump wasinserted into the peritoneal cavity; the muscle layer was sutured; and the skin incisionwas closed with wound clips or suturing.Autopsy and histopathology. Animals were autopsied, and all tissues were exam-ined regardless of their pathological status. Normal and tumour tissue samples werefixed in 10% neutral-buffered formalin (Sigma) overnight. Tissues were processedby ethanol dehydration and embedded in paraffin according to standard protocols.Sections (5 mm) were prepared for antibody detection and haematoxylin and eosinstaining. To evaluate evidence of invasion, sections were cut at 20-mm intervalsthrough the tissue and stained with haematoxylin and eosin. Slides were preparedcontaining three to five of these sections.Flow cytometry analysis. For phenotype analysis, the isolated cells were re-suspendedin PBS containing 1% FCS (Sigma-Aldrich) and were pre-incubated with a purifiedanti-mouse CD16/CD32 antibody (eBioscience) for 30 min at room temperature.The cells were then washed and stained for 15 min at room temperature with thefollowing anti-mouse monoclonal antibodies: CD45 eFluor 450 (clone 30-F11);Gr-1–PE (clone RB6-8C5); CD11b–APC (clone M1/70); F4/80 eFluor780 (cloneBM8); NK1.1–PE (clone PK13); and CD19–FITC (clone 6D5). All of the antibodieswere purchased from eBioscience. Samples were acquired on a FACSCanto II flowcytometer (BD Biosciences) after fixation with 1% formaldehyde (Sigma-Aldrich).When needed, cells were sorted from the prostate single-cell suspension using aFACSAria Cell Sorter (BD Biosciences) after staining with specific antibodies for30 min at 4 uC in PBS containing 1% FCS. Data were analysed using FlowJo soft-ware (Tree Star).Treatment of mice with CXCR2 antagonist and docetaxel. For Gr-11 myeloidcell depletion, 7-week-old Pten2/2 mice were intraperitoneally injected with a CXCR2antagonist (SB265610, 2 mg kg21 in sterile PBS; Tocris) once a day. For the combi-natorial treatment, docetaxel was intraperitoneally injected (10 mg kg21) once a weekfor 3 weeks. Animals were killed at 10 weeks of age, and prostate tissues were harvested.Quantitative PCR (qPCR). RNA isolation (QIAGEN) and TaqMan reverse tran-scriptase reactions (Applied Biosystems) were performed according to the manu-facturer’s instructions. qPCR reactions (SYBR Green system; Bio-Rad) for each samplewere conducted in triplicate. The primer sequences were obtained from PrimerBank(http://pga.mgh.harvard.edu/primerbank/index.html). Each value was normalizedto the Gapdh level as a reference. The primer sequences used were as follows: Pai1forward, 59-TTGAATCCCATAGCTGCTT-39; Pai1 reverse, 59-GACACGCCATAGGGAGAGA-39; p16Ink4a forward, 59-CGCAGGTTCTTGGTCACTGT-39; p16Ink4a

reverse, 59-TGTTCACGAAAGCCAGAGCG-39; Il6 forward, 59-TAGTCCTTCCTACCCCAATTT-39; Il6 reverse, 59-TTGGTCCTTAGCCACTCCTTC-39; Ccl2forward, 59-GTGGGGCGTTAAACTGCAT-39; Ccl2 reverse, 59-CAGGTCCCTGTCATGCTTCT-39; Tgfb forward, 59-CTCCCGTGGCTTCTAGTGC-39; Tgfbreverse, 59-GCCTTAGTTTGGACAGGATCTG-39; Il1ra forward, 59-CTGCACTTCCACAGTCCAGA-39; Il1ra reverse, 59-CTTAGCCCGCTTCAGCTCTTT-39;Il1a forward, 59-CGAAGACTACAGTTCTGCCAT-39; Il1a reverse, 59-ATATGTGATGCCCTGGTGGT-39; Gapdh forward, 59-AGGTCGGTGTGAACGGATTTG-39; and Gapdh reverse, 59-TGTAGACCATGTAGTTGAGGT-39.Genome-wide gene expression analysis. Total RNA was isolated from epithelialand myeloid cell populations using an miRNeasy Mini kit (QIAGEN) following themanufacturer instructions and was quantified using a NanoDrop ND-1000 Spec-trophotometer (NanoDrop Technologies). RNA quality was assessed using an Agilent2100 Bioanalyzer (Agilent Technologies). Gene expression profiling was carried outusing the one-colour labelling method. Labelling, hybridization, washing and slidescanning were performed following the manufacturer’s protocols. Briefly, equal

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amounts of total RNA (100 ng) were amplified, labelled with Cy3 and purified withspin columns. Labelled specimens (600 ng) were hybridized to SurePrint G3 MouseGE 8360K Gene Expression Microarrays (Agilent). After 17 h, slides were washedand scanned (G2505C scanner, Agilent Technologies).Gene expression data analysis. Images were analysed using Feature Extractionsoftware v10.7 (Agilent). Raw data elaboration was carried out with Bioconductor(http://www.bioconductor.org), using the R (v3.0.2) statistical environment. Back-ground correction was performed with the normexp method with an offset of 50,and quantile was used for between-array normalization. The LIMMA (LInear Modelsfor Microarray Analysis) package was then used to identify differentially expressedgenes, using the empirical Bayes method to compute a moderated t-statistic. Geneset enrichment analysis (GSEA; v2.07) was performed to examine the associationbetween predefined gene sets and gene expression profiles of selected samples.Survival curves. Differential survival between patient subgroups was plotted andcalculated using Kaplan–Meier curves. Patients were stratified based on IL1RA andCD33 score values. Briefly, scores were rank ordered and divided into seven percentiles

(from the lowest to the highest values). Such stratification showed significant dif-ferences in overall survival within The Cancer Genome Atlas Pan-Cancer analysisproject (log-rank test). The Pan-Cancer data set matrix and clinical information31

were downloaded from the UCSC Cancer Genomics Browser (https://genome-cancer.ucsc.edu/).Statistical analysis. Statistical analysis of the data was performed using a two-tailed,unpaired Student’s t-test. Values are expressed as mean 6 s.e.m. (*P , 0.05;**P ,0.01;***P , 0.001). Significant differences in survival curves were calculated usingthe log-rank test. Correlation analysis in TMA staining evaluation was conductedwith Fisher’s exact test, using the estimated percentage of positively stained cells asdetermined by a pathologist (M.S.).

30. Alimonti, A. et al. Subtle variations in Pten dose determine cancer susceptibility.Nature Genet. 42, 454–458 (2010).

31. The Cancer Genome Atlas Research Network. The Cancer GenomeAtlas Pan-Cancer analysis project. Nature Genet. 45, 1113–1120(2013).

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Extended Data Figure 1 | Gr-11 myeloid cells infiltrate senescent tumoursin Ptenpc2/2 mice. a, Representative SA-b-gal staining of prostate sectionsfrom 8-week-old Ptenpc1/1 and Ptenpc2/2 mice. Original magnification, 3400.b, Representative confocal immunofluorescence (IF) images showing stainingof the epithelial marker cytokeratin 18 (CK18) (grey) and the senescencemarker pHP1c (green) in prostate tumours from Ptenpc2/2 mice. Cells werecounterstained with the nuclear marker DAPI (blue). A Ptenpc1/1 prostatenegative for pHP1c staining is also shown (inset). c, Representative confocal IFimage showing proliferating epithelial cells (CK18, grey; Ki-67, red) andsenescent epithelial cells (cytokeratin 18, grey; pHP1c, green) in Ptenpc2/2

prostate lesions. Cells were counterstained with the nuclear marker DAPI(blue). The histogram shows the quantification of CK181pHP1c1Ki-672,CK181pHP1c2Ki-671 and CK181pHP1c1Ki-671 cells (n 5 3; 1 tumourper mouse; 10 fields acquired; 412 cells counted). d, Quantification ofCD11b1Gr-11 immune cells in 8-week-old Ptenpc1/1 and Ptenpc2/2 mice(n 5 6). e, Flow cytometry analysis showing the heterogeneity of thetumour-infiltrating CD451CD11b1Gr-11 immune cells in Ptenpc2/2

prostates. f, In Ptenpc2/2 tumours, the senescence response starts at 8 weeksof age (top). A time course experiment is shown, indicating the recruitmentof Gr-11 myeloid cells in Ptenpc1/2 and Ptenpc2/2 mice at the onset oftumorigenesis (bottom) (n 5 3 per group; 1 tumour per mouse). g, Correlationbetween Ki-67 staining and percentage of Gr-11 myeloid cells in the anterior(AP) and dorsolateral lobes (DLP) of Ptenpc2/2 tumours (n 5 3; 1 tumourper mouse). h, Experimental scheme. Ptenpc2/2 mice were lethally irradiatedand then transferred with bone marrow from UBC-GFP mice that had beendepleted of T-, B- and natural killer (NK) cells. Prostate tissues were collected

4 weeks after transfer. i, Representative confocal IF images showing thelocalization of myeloid cells (green) infiltrating the anterior prostate glandof Ptenpc2/2UBC-GFP mice. Proliferating cells (Ki-67, red) and stroma(vimentin, grey) are also shown. Cells were counterstained with DAPI (blue).j, Representative confocal IF image showing the localization of tumour-infiltrating UBC-GFP cells and proliferating epithelial cells (CK18, grey;Ki-67, red) in prostate lesions from Ptenpc2/2UBC-GFP mice. Cells werecounterstained with the nuclear marker DAPI (blue). Arrows indicateCK181Ki-671 cells, which were considered for the analysis, while * indicatesCK182Ki-671 cells, which were excluded from the analysis. k, Quantificationof UBC-GFP1Gr-11 cells (n 5 4; 1 tumour per mouse; 5 fields acquired;300 cells counted). l, Representative confocal IF image showing the localizationof tumour-infiltrating myeloid cells (Gr-1, red) and proliferating epithelial cells(CK18, grey; Ki-67, green) in prostate lesions from non-irradiated Ptenpc2/2

mice. Cells were counterstained with the nuclear marker DAPI (blue). Thehistogram shows the quantification of the distance between tumour-infiltratingGr-11CK182 myeloid cells and CK181Ki-671 proliferating epithelial cells(n 5 3; 10 fields acquired; 334 measurements). The arrows indicate CK181Ki-671 cells, which were considered for the analysis. m, Experimental set-up.n, Flow cytometry and qRT–PCR analysis of Gr-11 myeloid cells differentiatedin vitro in the presence of granulocyte–macrophage colony-stimulatingfactor (GM-CSF) and IL-6, showing upregulation of Gr-1 and Il10 mRNA.o, SA-b-gal staining of Pten2/2 MEFs. c, d, f, g, k, l, n, Error bars, mean 6 s.e.m.P values were derived from an unpaired, two-tailed Student’s t-test(**P , 0.01; ***P , 0.001).

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Extended Data Figure 2 | Gene expression analysis of factors expressed byGr-11 myeloid cells and epithelial cells sorted from Ptenpc2/2 tumours.a, Experimental set-up (top). Protein levels of IL-1RA in Ptenpc2/2 bulkprostate tumours and Ptenpc2/2 immunodepleted prostatic epithelial cells(bottom) (n 5 3 per group; 1 tumour per mouse). b, Experimental set-up. Geneexpression analysis of epithelial cells and Gr-11 myeloid cells purified fromPtenpc2/2 prostate tumours. Briefly, prostates were isolated from 8-week-oldPtenpc2/2 mice and processed to a single-cell suspension. CD452 epithelialcells and CD451CD11b1Gr-11 myeloid cells were further sorted using a

FACSAria Cell Sorter. Total RNA was isolated from the epithelial and myeloidcell populations, and gene expression profiling was carried out using theone-colour labelling method, performing two replicates for each condition.A heatmap displaying the mRNA expression of 53 secreted factors is shown(n 5 2 per group). c, qRT–PCR analysis of CD11b1Gr-12F4/801 sorted fromPtenpc2/2 prostate tumours, showing Il1ra expression (n 5 3). a, c, Error bars,mean 6 s.e.m. P values were derived from an unpaired, two-tailed Student’st-test (**P , 0.01; ***P , 0.001).

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Extended Data Figure 3 | Gr-11 myeloid cells oppose senescence in bothPten-loss-induced cellular senescence and oncogene-induced senescence.a, Western blot analysis showing the inverse correlation between IL-1RA andp16INK4A protein levels in the anterior (AP) and dorsolateral lobes (DLP) ofPtenpc2/2 prostate tumours. Levels are normalized to E-cadherin expression.b, Il1ra mRNA expression of bone marrow precursors (BMPs) and Gr-11

myeloid cells sorted from Ptenpc2/2 prostate tumours (n 5 3). c, Cell growth ofH-ras MEFs cultured in the presence of conditioned medium from Gr-11

myeloid cells (n 5 3). d, Quantification (left) and representative images (right)of SA-b-gal1 H-ras MEFs cultured in the presence of conditioned medium

from Gr-11 myeloid cells (n 5 3). e, Il1ra mRNA expression of Gr-11

myeloid cells differentiated for 4 days with IL-6 and GM-CSF, in the absence orpresence of the JAK2 inhibitor NVP-BSK805, compared with bone marrowprecursors (BMPs) (n 5 3). f, Quantification (left) and representative images(right) of SA-b-gal1 Pten2/2 MEFs cultured in the presence of conditionedmedium from Gr-11 myeloid cells that had been pre-treated with the JAK2inhibitor NVP-BSK805 (n 5 3). a–f, Error bars, mean 6 s.e.m. P valueswere derived from an unpaired, two-tailed Student’s t-test (**P , 0.01;***P , 0.001).

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Extended Data Figure 4 | IL-1RA opposes docetaxel-induced senescence inLNCaP cancer cells in vitro. a, Histogram showing the quantification ofpHP1c1 cells. Briefly, LNCaP prostate cancer cells were cultured in the absenceor presence of docetaxel, with or without human recombinant IL-1RA. After5 days, cells were collected and stained for immunofluorescence analysis.Representative confocal IF staining showing senescent pHP1c1 (red) LNCaP

cancer cells (inset). Cells were counterstained with the nuclear marker DAPI(blue). b, Cell growth of LNCaP cells cultured in the absence or presence ofdocetaxel, with or without human recombinant IL-1RA (n 5 3). a, b, Errorbars, mean 6 s.e.m. P values were derived from an unpaired, two-tailedStudent’s t-test (*P , 0.01; ***P , 0.001).

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Extended Data Figure 5 | Gr-11 myeloid cells infiltrate adenocarcinomaareas in lungs from K-ras1/G12V mice. a, Haematoxylin and eosin (H&E),p16INK4A, p19ARF, Gr-1, IL-1RA and Ki-67 immunohistochemical staining inlungs from K-ras1/G12V mice. Original magnification, 3400. Staining of bothadenocarcinoma and adenoma areas is shown (left). Histograms showingquantification of cells positive for p16INK4A, p19ARF, Gr-1, IL-1RA and Ki-67(right) (n 5 7; 3 sections per mouse; $5 fields per section analysed).b, c, Representative confocal IF images showing staining of Ki-67 (green)

and the myeloid marker Gr-1 (red) with (b) or without (c) the epithelial markerCK18 (grey), in adenocarcinoma and adenoma areas of lungs from K-rasG12V

mice. Cells were counterstained with the nuclear marker DAPI (blue).Panel b magnification 3 400. Panel c magnification 3 200. Top panel insets inc show H&E staining (magnification 3100) of the same areas stained for IF;bottom panels, magnification 3 400. a, Error bars, mean 6 s.e.m. P valueswere derived from an unpaired, two-tailed Student’s t-test (*P , 0.05;**P , 0.01; ***P , 0.001).

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Extended Data Figure 6 | Senescence and apoptotic markers inPtenpc1/1Il1ra1/1 and Ptenpc2/2Il1ra2/2 mice. a, mRNA levels of Il1rain BMPs from the indicated genotypes (n 5 3). b, Quantification of Ki-67staining. c, d, p16INK4A mRNA and protein levels. e, PAI1 mRNA and proteinlevels. f, SA-b-gal staining in prostate tissues from Ptenpc1/1Il1ra1/1 andPtenpc2/2Il1ra2/2 mice (n 5 4 mice per group; 1 tumour per mouse;3 sections per tumour; $5 fields per section). g, Ki-67 and p16INK4A

immunohistochemical staining of stage-matched prostate tumours from

Ptenpc1/1Il1ra1/1 and Ptenpc2/2Il1ra2/2 mice. The histograms showquantification of Ki-67 and p16INK4A positivity (n 5 4 mice per group;1 tumour per mouse; 3 sections per tumour; 3 fields per section).h, Immunohistochemistry for cleaved caspase-3 in prostate tissues fromPtenpc1/1Il1ra1/1 and Ptenpc2/2Il1ra2/2 mice. a–e, g, h, Error bars,mean 6 s.e.m. P values were derived from an unpaired, two-tailed Student’st-test (*P , 0.05; **P , 0.01; ***P , 0.001).

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Extended Data Figure 7 | Efficiency of magnetic-activated cell sorting(MACS) purification and bone marrow transplantation. a, Representativeflow cytometry plots showing whole bone marrow cells, phycoerythrin (PE)-positive cells isolated after magnetic separation and bone marrow cells depletedof T, NK and B cells before adoptive transfer to irradiated Ptenpc2/2 mice(gating on total cells). Briefly, cells were flushed from the long bones of donor

mice and stained with the following anti-mouse antibodies: anti-CD3–PE,anti-NK1.1–PE and anti-CD19–PE. The cells were then washed and stainedwith anti-PE magnetic beads and collected for magnetic separation.b, Representative plots obtained from flow cytometry analysis of splenocytesisolated from Ptenpc2/2 mice before and after lethal irradiation (top). Immunereconstitution 14 days after bone marrow transplantation (bottom).

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Extended Data Figure 8 | Effect of IL1-a and CXCR2a on Ptenpc2/2

tumours. a, Experimental scheme. Briefly, osmotic pumps were implantedin the peritoneal cavity of six Ptenpc2/2 mice, to expose the prostate tissueto a continuous and controlled concentration of either IL-1a or PBS.b, Immunohistochemical staining (H&E, Ki-67 and p16INK4A) of prostatesections from Ptenpc2/2 mice treated with IL-1a or PBS. c–e, Histogramsshowing quantification of glands affected by prostatic intraepithelial neoplasia(PIN) (c), Ki-67 positivity (d) and p16INK4A positivity (e) (n 5 3 per group;1 tumour per mouse; 3 sections per mouse; 3 fields per sections were analysed).f, Protein profile of immunodepleted epithelial cells showing the high levels ofcytokines that recruit (CXCL1 and CXCL2) and activate (GM-CSF and IL-6)Gr-11 myeloid cells in Ptenpc2/2 prostate tumours. g, Experimental set-up.

Doce, docetaxel. h, Flow cytometry plots showing the reduced recruitment ofGr-11 myeloid cells in Ptenpc2/2 mice after treatment with a CXCR2antagonist (CXCR2a), with gating on live CD451 cells. The histogram showsthe frequency of Gr-11 myeloid cells (n 5 5 control group; n 5 7 treatedgroups). i, j, Flow cytometry plots showing the recruitment of Gr-11 myeloidcells to the peripheral lymph nodes (upper panels) and spleen (lower panel)isolated from Ptenpc2/2 mice, after treatment with CXCR2a, with gating onlive cells. The histograms (right) show the frequency of Gr-11 myeloid cellsin the lymph nodes and spleen from Ptenpc2/2 mice, after treatment withCXCR2a (n 5 5 mice per group). c–f, h–j, Error bars, mean 6 s.e.m. P valueswere derived from an unpaired, two-tailed Student’s t-test (*P , 0.05;**P , 0.01; ***P , 0.001).

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Extended Data Figure 9 | Treatment with a CXCR2 antagonist in vivo.a, Il1ra mRNA levels in Ptenpc2/2 tumours after treatment with CXCR2a aloneor in combination with docetaxel. b, Immunohistochemical staining (H&E andSA-b-gal) in mice treated with CXCR2a and docetaxel. c, Quantification ofcleaved caspase-3 in Ptenpc2/2 tumours after the indicated treatments.d, Immunohistochemical staining for Ki-67 in stage-matched prostate tumoursfrom Ptenpc2/2 mice after treatment. The histograms show quantification

of Ki-67 and p16INK4A positivity. e, f, Treatment of Pten2/2 MEFs withCXCR2a (n 5 3). NS, not significant. g, Staining and quantification of IL-1RAin primary tumours from patients. Responder patients (‘‘R’’) and non-responder patients (‘‘NR’’), based on disease-free survival. a, c–e, Error bars,mean 6 s.e.m. P values were derived from an unpaired, two-tailed Student’st-test (*P , 0.05; **P , 0.01). b–d, Control n 5 5; treated n 5 7; 3 sections permouse; 5 fields per section.

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Extended Data Figure 10 | Evidence in human samples and proposedmodel. a, b, Graphs showing the inverse association between p16INK4A andCD33 in the tissue microarrays and single prostate sections from humanprostate cancer. Box plots in a show the interquartile range, whiskers show thefull range. c, Histogram showing the percentage of cases positive forKi-67, p16INK4A and CD33 in sections. Normal-like prostate areas werecompared with PIN and prostate cancer (PCA) areas in the same section.d, Kaplan–Meier analysis (see the Survival curves subsection in Methods).

e, Gr-11 myeloid cells recruited to the tumour site oppose Pten-loss-inducedcellular senescence by secreting IL-1RA in the tumour microenvironment.f, Gr-11 myeloid cells can protect tumour cells from senescence by tiltingthe balance between IL-1a and IL-1RA in the tumour microenvironment.Pharmacological interventions aimed at impairing Gr-11 myeloid cellrecruitment (for example, CXCR2a) can enhance senescence, thus improvingchemotherapy efficacy. CIS, chemotherapy induced senescence. a, Correlationassessed with Fisher’s exact test.

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