blocking il1b pathway following paclitaxel chemotherapy ...number of m2 macrophages and vessel...

Cancer Biology and Signal Transduction

Blocking IL1b Pathway Following PaclitaxelChemotherapy Slightly Inhibits Primary TumorGrowth but Promotes Spontaneous MetastasisTali Voloshin1, Dror Alishekevitz1, Limor Kaneti2, Valeria Miller1, Elina Isakov3,Irena Kaplanov4, Elena Voronov4, Ella Fremder1, Moran Benhar3, Marcelle Machluf2,Ron N. Apte4, and Yuval Shaked1

Abstract

Acquired resistance to therapy is a major obstacle in clinicaloncology, and little is known about the contributing mechan-isms of the host response to therapy. Here, we show that theproinflammatory cytokine IL1b is overexpressed in responseto paclitaxel chemotherapy in macrophages, subsequentlypromoting the invasive properties of malignant cells. In accor-dance, blocking IL1b, or its receptor, using either genetic orpharmacologic approach, results in slight retardation of pri-mary tumor growth; however, it accelerates metastasis spread.Tumors from mice treated with combined therapy of paclitaxel

and the IL1 receptor antagonist anakinra exhibit increasednumber of M2 macrophages and vessel leakiness when com-pared with paclitaxel monotherapy-treated mice, indicating aprometastatic role of M2 macrophages in the IL1b-deprivedmicroenvironment. Taken together, these findings demonstratethe dual effects of blocking the IL1 pathway on tumor growth.Accordingly, treatments using "add-on" drugs to conventionaltherapy should be investigated in appropriate tumor modelsconsisting of primary tumors and their metastases. Mol CancerTher; 14(6); 1385–94. �2015 AACR.

IntroductionChemotherapy is commonly used for the treatment of several

malignancies. Although treatment efficacy is usually achieved,tumor relapse andmetastasis spread are often observed (1). Thus,ongoing efforts are being made to overcome the resistance ofcancer cells to chemotherapy, and to prevent metastases.

In recent years, some of these efforts are directed towardunderstanding the diverse and dual nature of the tumor micro-environment, as manifested in response to anticancer treatments.For instance, preclinical studies demonstrated that on one hand,chemotherapy supports the secretion of various protumorigenicfactors from macrophages and myeloid-derived suppressor cells(MDSC; refs. 2, 3), and on the other, it acts against tumor cells byinducing the immune cell response (4, 5). In addition to the localtumor microenvironment, the host response to chemotherapymay also promote tumor regrowth and metastasis by differentmeans, including rapid mobilization and tumor homing of

different bone marrow-derived cells (BMDC; ref. 6) and upregu-lation of proangiogenic and prometastatic factors (7). As such,preclinical and clinical testing of promising add-on therapies thattarget the induction of host response components provide amplesupport for their use as novel antitumor strategies (8).

The inflammatory cytokine IL1b has been studied in a consid-erable number of malignancies. Its correlation with poor prog-nosis in patients with cancer has established a ground for theidentification of its proangiogenic and prometastatic role incancer (9). The induction of IL1b expression in the spleen ofmice treated with 5-FU and gemcitabine chemotherapies has ledto studying whether targeting its pathway can improve therapyoutcome (3). Indeed, the IL1R antagonist (IL1Ra, anakinra),which is approved for the treatment of rheumatoid arthritis inpatients (10), was shown to abrogate the protumorigenic effect ofnaturally occurring and posttherapy-induced IL1b (3). However,although the prometastatic role of IL1b is bona fide and repeat-edly reported in various preclinical models, the actual conse-quence of postchemotherapy-induced IL1b blockade has not yetbeen fully explored.

Here, we show that IL1b levels are elevated in response topaclitaxel chemotherapy. Blocking IL1b following paclitaxelslightly inhibits the growth of primary tumors; however, itspotential as a therapeutic strategy for cancer treatment is doubtful,since it is shown to increase spontaneous metastatic spread.

Materials and MethodsMice

Six- to 8-week-old C57Bl/6 IL1b�/� knockout mice and theirwild-type counterparts (fromBen-GurionUniversity of theNegev,Israel), C57Bl/6, and BALB/c (Harlan) were used in experimentalprotocols approvedby theAnimal Care andUseCommittee of theTechnion-Israel Institute of Technology (Haifa, Israel).

1DepartmentofCell BiologyandCancerScience,RappaportFacultyofMedicine, Technion, Haifa, Israel. 2Faculty of Biotechnology,Technion,Haifa, Israel. 3Department of Biochemistry, Rappaport Faculty ofMedicine, Technion, Haifa, Israel. 4The Shraga Segal Department ofMicrobiology, Immunonology and Genetics, Faculty of HealthSciences, Ben-Gurion University of the Negev, Beer Sheva, Israel.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Yuval Shaked, Department of Cell Biology and CancerScience, Rappaport Faculty of Medicine, Technion–Israel Institute of Technol-ogy, 1 EfronSt. BatGalim, Haifa 31096, Israel. Office: Phone: 972-4-829-5215; Fax:972-4-855-5221; E-mail: [email protected].

doi: 10.1158/1535-7163.MCT-14-0969

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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Published OnlineFirst April 17, 2015; DOI: 10.1158/1535-7163.MCT-14-0969

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Tumor cell linesLewis lung carcinoma (LLC) and MDA-MB-231 human breast

carcinoma (obtained from the ATCC) were cultured in DMEMsupplemented with10% (v/v) FCS, 100 U/mL penicillin, 100 mg/mL streptomycin, 2mmol/L L-glutamine, and 4mmol/LHEPES at37�C in a humidified atmosphere containing 5% CO2. 4T1murine mammary adenocarcinoma cancer cells (ATCC) werecultured in RPMI-1640 supplemented with10% (v/v) FCS, 100U/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L L-glutamine at 37�C in a humidified atmosphere containing 5%CO2. All cells were obtained between 2008 and 2010, and theywere passed in culture for no more than 4 months after beingthawed from authentic stocks. Cell lines were routinely tested formycoplasma contamination.

Drug treatment protocolFor in vivo studies, paclitaxel chemotherapy (25 mg/kg for

BALB/c or 50 mg/kg for C57Bl/6 mice) was administeredintraperitoneally as a single bolus injection, in doses previ-ously described (6). Anakinra (Kineret, Biovitrum; 10 mg/kg)was injected intraperitoneally daily, starting one day beforechemotherapy administration, and as indicated in the text.Control mice were administered with the relevant vehicles. Forin vitro studies, cells were exposed to 200 nmol/L paclitaxelchemotherapy or 0 to 100 ng/mL anakinra. In some experi-ments, depletion of macrophages was achieved by using clo-dronate-containing liposomes (clodrolip), as previously pub-lished (11) and as detailed in Supporting Information Online.The liposomes were injected intraperitoneally to mice with0.15 mL of either control Sham or clodrolip, at a concentrationof 3 to 3.5 mg/mL, 48 hours before injection of paclitaxelchemotherapy.

Tumor implantation4T1 cells (5�105 cells) were orthotopically injected in 50 mL

volumes into the inguinal mammary fat pad of BALB/c mice.LLC cells (5�105 cells) were subcutaneously injected in thehind flank of 8-week-old C57Bl/6 mice. The tumor size wasassessed regularly with Vernier calipers using the formulawidth2 � length �0.5. Treatment was initiated when tumorsreached either 200 mm3 or 500 mm3 as indicated in the text.Mice were randomly grouped (n ¼ 5–7/group, as specified inthe text) before treatment with paclitaxel, anakinra, paclitaxel,and anakinra, or vehicle control. For an experimental metas-tasis model, LLC cells (2.5�104) stably transfected with plas-mid for cytoplasmic GFP expression, were intravenouslyinjected via the tail vein (n ¼ 4–6 mice/group). Mouse survivalwas monitored.

Bone marrow transplantationBone marrow transplantation was performed as previously

described (6, 7). Briefly, bone marrow cells were obtained byflushing femurs and tibias of 8-week-old donor C57Bl/6 WT andC57Bl/6 IL1b�/� mice. The bone marrow cells (5�106 per recip-ient) were transplanted by tail vein injection into lethally irradi-ated C57Bl/6 WT recipients (1,000 cGy total body irradiation(250 cGy/minute) using Elekta Precise (ElektaOncology Systems)linear accelerator 6MeV photon beam radiation (Department ofRadiation Therapy, Rambam Medical center, Haifa, Israel).Experiments were performed 4 to 6 weeks after bone marrowcell reconstitution.

Macrophage isolation and cultureEight-week-old BALB/c mice were injected intraperitoneally

with 3 mL 4% thioglycollate solution (BD Biosciences). Threedays later, animals were euthanized and peritoneal lavage wasperformed. Peritoneal exudate cells (5�106 cells) were plated in60-mm dish (Corning) for 24 hours and cultured in RPMIcomplemented with 10% FCS, 100 U/mL penicillin, 100 mg/mLstreptomycin, 2 mmol/L L-glutamine, and 4 mmol/L HEPES.Nonadherent cells were removed after 24 hours by washing. Formacrophage stimulation, cells were incubated with mediumsupplemented with 1 mmol/L arginine and LPS (0.5 mg/mL) for4 hours following one hour exposure to ATP (5 mmol/L) orpaclitaxel (200 nmol/L). In some experiments,macrophages werecultured in the presence of recombinantmurine IL1b (1 pg, 10 pg,or 100 pg, Peproteck). All experiments were performed intriplicates.

Modified Boyden chamber assayInvasion assay was performed using modified Boyden cham-

bers as previously described (7). Briefly, filters (6.5 mm indiameter, 8 mm pore size) were coated with Matrigel (BD Bios-ceinces) and dried for 2 hours at 37�C. Serum-deprived LLC andMDA-MB-231 cells (2�105) were seeded on the upper compart-ment of the chamber. The lower compartment was filled withserum-free DMEM that contained 10% plasma from non–tumor-bearing mice that were treated for 24 hours with paclitaxel,anakinra, combined treatment of paclitaxel and anakinra,or vehicle control; with plasma obtained from WT IL1b orIL1b�/� bone marrow transplanted mice; with peritoneal macro-phages, recombinant IL1b (in escalating concentrations), or thecombination of the two; or with plasma depleted from IL1b usinga specific neutralizing antibody (15mg; R&D systems) followed byimmunoprecipitation and depletion using Protein A/G mixedSepharose beads. After incubation for 6 and 18 hours at 37�C in a5%CO2 incubator, the cells that invaded to the bottomfilter, werestained with 0.5% crystal violet (Sigma) and counted under aninverted microscope (Lieca DMIL LED) per �100 objective field.All in vitro Boyden chamber experiments were performed intriplicates.

ELISANon–tumor-bearing paclitaxel-treated and control C57Bl/6

and BALB/c mice were euthanized by isoflorane inhalation andblood was collected via cardiac puncture. For BALB/c mice,organs such as brain, liver, lungs, kidney, spleen, bone marrow,and blood were obtained from the mice at several time pointsafter they were treated with paclitaxel or vehicle control (n ¼ 5mice/group). Organs were homogenized in PBS containing 20mmol/L HEPES, 100 mmol/L NaCl, 1 mmol/L EDTA, 1%Triton, and a protease inhibitor mixture (Roche Diagnostics).After lysate centrifugation, supernatant was collected. In sepa-rate experiments, a total of 200 to 300 mm3 of LLC tumor tissue(n ¼ 5–6 samples/group) or cultured peritoneal macrophageswere collected and homogenized as described above. Equalamounts of protein were applied on a mouse IL1b ELISA (R&DSystems, Inc.) in accordance with the manufacturer's instruc-tions. For detection of IL1b levels in plasma, pooled plasmasamples were concentrated through Amicon ultra-0.5 centrif-ugal filter device (at 7–10-fold) before use in order to reach thedetection threshold. All experiments detecting IL1b expressionlevels were performed in triplicates.

Voloshin et al.

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Flow-cytometric analysisM1 and M2 macrophages were quantitated using flow cyto-

metry as described (6, 12). Briefly, tumors and lungs from treatedand nontreated mice were collected and prepared as single-cellsuspensions as previously described (6, 13). M1 macrophageswere identified as F4/80þ, CD11cþ, CD206� CD45þ; and M2macrophages were identified as F4/80þ, CD11c�, CD206þ,CD45þ, using BioLegend and BD Biosciences monoclonal anti-bodies conjugated with fluorescent dyes as follows: F4/80- Phy-coerythrin (PE); CD11c-Peridinin Chlorophyll Protein (PerCP);CD206-Allophycocyanin (APC); CD45-APC-Cyanin-7 (APC-Cy7). For clodronate liposomes-treated mice, the verification ofmacrophage depletion was performed using the F4/80 surfacemarker. Flow-cytometric experiments were performed on Cyan-ADP flow cytometer (Beckman Coulter) and analyzed with Sum-mit Version 4.3 (Beckman Coulter) and FlowJo Version 8 (Ash-land) flow-cytometric analysis software.

Quantitation and visualization of tissuesTissue processing was performed as previously described (6).

Briefly, tissue cryosections (10 mm) were used to analyze bloodvessel perfusion by the DNA-binding dye Hoechst 33342 (40mg/kg; Sigma-Aldrich) or lung metastasis using hematoxylin andeosin (H&E) or DAPI. Vessels were immunostained with endo-thelial cell-specific antibody (anti-CD31, 1:200 ratio, BD Bios-ciences), and the number of vessel structures per field werecounted and plotted (5 fields/tumor; n > 20 fields per group).Metastatic lesions were counted per field, and GFPþ lung metas-tases were analyzed by flow cytometry as described above. Sec-tions were visualized under a Leica CTR 6000 microscope (LeicaMicrosystems) using Leica Application Suite Version 3 or the3DHISTECH Panoramic MIDI system using a HITACHI HV-F22 color camera and the Panoramic Viewer visualization andanalysis software.

Gene expression analysis by RT-PCRRNAwas extracted from spleen or blood obtained frommice as

indicated in the text, using the RNeasy Mini Kit (QIAGEN), andsubsequently converted into cDNA by reverse transcriptase (Pro-mega). The gene expression of mouse IL1b was determined by7000 Real-Time PCR system (Applied Biosystems). For moredetails, see Supporting Information Online.

Statistical analysisData are expressed asmean� SD, and the statistical significance

of differences was assessed by one-way ANOVA, followed byNewman–Keuls ad hoc statistical test using GraphPad Prism 4software (GraphPad Software, Inc.). Differences between allgroups were compared with each other, and were consideredsignificant at values of �, P < 0.05; ��, P < 0.01; and ��, P < 0.001.

ResultsHost response to paclitaxel chemotherapy contributes to tumorcell invasiveness in an IL1-IL1R–dependent manner

We previously demonstrated that paclitaxel chemotherapyadministered to non–tumor-bearingmice induces the productionof systemic factors that promote tumor cell migration, invasion,and metastasis (7). In order to identify such factors and todistinguish between host and tumor cell-derived effects followingchemotherapy, we screened plasma samples from non–tumor-bearing C57Bl/6 and BALB/c mice administered with paclitaxel.IL1b levels were significantly elevated 24 hours following pacli-taxel administration (Fig. 1A). This induction in IL1b levels wasblocked by concomitant administration of paclitaxel and ana-kinra (Supplementary Fig. S1A). Interestingly, levels of IL1b inplasma from anakinra-treated mice were undetectable, but thiswas not due to the direct effect of anakinra on detection of IL1b byELISA (data not shown). Of note, levels of matrix metalloprotei-nase-9 (MMP9) were substantially increased in the plasma from

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Figure 1.Paclitaxel induces host-dependentIL1b production. A and B, eight- to10-week-old non–tumor-bearingC57Bl/6 and BALB/c mice weretreated with vehicle (control) orpaclitaxel (PTX) chemotherapy(n ¼ 5 mice/group). After 24 hours,mice were sacrificed and the level ofIL1b from pooled plasma (A) andfrom various organs (for BALB/c; B)was evaluated by ELISA. C, BALB/cmice were treated with vehicle orpaclitaxel (n ¼ 5 mice/group) andspleens were removed at 0, 6, 12, 24,and 48 hours posttreatment for theassessment of IL1b expression byELISA. D, relative IL1b transcriptlevels in spleens of C57Bl/6 andBALB/c mice treated with paclitaxelfor 24 hours were assessed byquantitative RT-PCR. HPRT servedas the internal control. (n ¼ 3repeats/group). Error bars, � SD.� , P < 0.05; �� , P < 0.01; �� , P < 0.001.

Dual Role for IL1b in Tumors

www.aacrjournals.org Mol Cancer Ther; 14(6) June 2015 1387




paclitaxel-treatedmicewhen comparedwith their levels in controlmice, similar to a previous report (7); however, MMP9 levels weredecreased in plasma from mice treated with paclitaxelþanakinra(Supplementary Fig. S1B). Focusing on BALB/c mice, the analysisof IL1b levels in different organs revealed that the spleen was theexclusive organ exhibiting a significant increase in IL1b levelsfollowing paclitaxel therapy (Fig. 1B), similar to gemcitabine and5-FU chemotherapies as previously reported (3). IL1b levels in thespleen but not in other organs peaked at 12 hours and remainedsignificantly high for at least 48hours (Fig. 1C and SupplementaryFig. S1C). These results were confirmed by analyzing IL1bmRNAexpression in the spleen 24 hours following paclitaxel adminis-tration. However, IL1b mRNA expression levels were loweredwhen paclitaxel was administered in combination with anakinra.Comparable results were observed when C57Bl/6 mice weretreated with paclitaxel as assessed by RT-PCR (Fig. 1D andSupplementary Fig. S1D). To directly assess the contribution oftherapy-induced, host-derived IL1b to the promotion of tumorcell invasiveness as we reported in previous study (7), we per-formed a modified Boyden chamber assay in the presence of

plasma obtained from non–tumor-bearing mice treated withpaclitaxel, anakinra, or the combination of the two drugs at 6,24, and 48 hours posttreatment. Plasma from paclitaxel-treatedmice induced LLC and MDA-MB-231 tumor cell invasion, aneffect that was significantly blocked when paclitaxel treatmentwas combined with anakinra (Fig. 2). Of note, an increase ininvasion properties of tumor cells was also demonstrated in aprevious study where tumor cells were exposed to plasma fromLLC tumor–bearing mice treated with paclitaxel chemotherapy(7). We verified that neither anakinra nor IL1b-depleted plasmadirectly affects tumor cell invasion (Supplementary Fig. S1E).Consistently, the motility of MDA-MB-231 cells was greater inthe presence of plasma from non–tumor-bearing paclitaxel-treated mice, when compared with plasma from vehicle, ana-kinra, or paclitaxelþanakinra-treated mice as assessed bywound scratch assay (Supplementary Fig. S1F). We should notethat the motility assay could not be performed on LLC cells dueto their growth pattern in culture. Overall, these results dem-onstrate that paclitaxel induces host-dependent secretion ofIL1b, which may account for cancer cell invasion properties.

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Figure 2.Anakinra blocks invasive paclitaxel-induced tumor cell invasion in vitro. Eight- to 10-week-old non–tumor-bearing BALB/c mice were treated with vehicle (control),paclitaxel, anakinra, or the combination of paclitaxel and anakinra. Plasma was extracted at 6, 24, and 48 hours after treatment administration. A, invasionproperties of murine LLC and MDA-MB-231 human breast cancer cells were evaluated by the modified Boyden chamber assay in the presence of the plasmasamples (magnification, �100; scale bar, 200 mm). B, quantification of invading cells was performed using ImageJ software (NIH). Error bars, � SD. � , P < 0.05;�� , P < 0.01; �� , P < 0.001.

Voloshin et al.





Paclitaxel stimulates macrophages to secrete IL1b promotingthe invasive properties of cancer cells

Macrophages have been described as a major source of IL1b,which is produced and secreted by the inflammasome-mediatinginflammatory response (14, 15). Therefore, we first assessed theeffects of paclitaxel on macrophage IL1b synthesis and secretion,in order to explain the elevation in IL1b levels in plasma andspleen following paclitaxel treatment. Previous work has indicat-ed that paclitaxel increases the production of IL1b precursor inmonocytes, but not its secretion (16). Indeed, in vitro stimulationof macrophages with a single treatment of paclitaxel resulted in

production of IL1b within macrophages, which was not furtherincreased following activation with LPS, a proinflammatory stim-ulator, as evaluated inmacrophage lysates (Fig. 3A). However, theincrease in IL1b production did not result in IL1b secretion fromthe paclitaxel-treated macrophages as compared with macro-phages stimulated with LPS in combination with ATP, as evalu-ated in the conditioned medium of macrophages (Fig. 3B). Theseresults suggest that in the in vitro system used here, macrophages,on their own, do not secrete IL1b in response to paclitaxel due tothe absence of additional stimuli that are otherwisemaypresent invivo.We therefore investigated the in vivoproduction and secretion

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Figure 3.Paclitaxel promotes secretion of IL1b from macrophages. A and B, peritoneal macrophages obtained from 8-week-old BALB/c mice (n ¼ 4 mice/group) weretreated with vehicle control or paclitaxel, or stimulated for 4 hours with LPS alone or following one hour exposure to ATP or paclitaxel. The level of IL1b in themacrophage lysate (A) or medium from cultured macrophages (B) was assessed by ELISA. C, eight-week-old BALB/c mice underwent macrophage depletionusing clodronate-containing liposomes (clodrolip) as described in Materials and Methods. Sham liposome injection was set as control (n ¼ 5 mice/group).Forty-eight hours later, the mice were treated with vehicle (control) or paclitaxel (PTX). After 24 hours, mice were sacrificed, plasma was pooled, and spleens wereremoved and evaluated for IL1b expression using ELISA. The experiment was repeated three times on pooled plasma samples. D, the invasive properties ofLLCandMDA-MB-231 cellswere evaluatedusing themodifiedBoyden chamber assay,with different concentrations of recombinant IL1b in the presenceor absence ofmacrophages cultured in the lower chamber (magnification �100, scale bar, 200 mm). Quantification of cells/field is shown in Supplementary Fig. S2D.Error bars, � SD. �, P < 0.05; �� , P < 0.001.






of IL1b in response to paclitaxel. To do so, macrophages weredepleted from BALB/c mice using Clodronate-containing lipo-somes (Clodrolip) as confirmed by flow cytometry of the spleens(Supplementary Fig. S2A). The mice were then treated withvehicle control or paclitaxel and the level of IL1b in the spleenwas assessed by ELISA. Splenic IL1b levels following paclitaxeltreatmentwere significantly higher in sham liposome-treatedmicewhen compared with Clodrolip-treated (macrophage-depleted)mice in which their IL1b levels were not significantly differentfrom untreated control mice. Comparable results were observedin IL1b levels in the plasma of mice treated with Clodrolip (Fig.3C). Moreover, the depletion of macrophages abrogated theproinvasive properties of plasma from paclitaxel-treated mice(Supplementary Fig. S2B). Collectively, these findings suggestthat macrophages are a major source of paclitaxel-induced IL1b,at least in the spleen, and the production and secretion of IL1b bymacrophages in response to paclitaxel promote cancer cell inva-sive properties.

We next asked whether IL1b acts directly on malignant cells topromote their invasiveness. Coincubation of LLC and MDA-MB-231 cells with a high dose (100 pg) of recombinant IL1b did notalter the invasive properties of these cells as assessed by themodified Boyden chamber assay (Fig. 3D), despite the expressionof the IL1 receptor type I (IL1RI) in these cell lines as assessed byflow cytometry (Supplementary Fig. S2C). These results suggestthat paclitaxel-induced IL1b possibly acts on the host to promotetumor cell invasion rather than directly on the malignant cells.Indeed, when macrophages where incubated in the lower com-partment of the Boyden chamber along with escalating doses ofIL1b, the invasive properties of both LLC and MDA-MB-231 cellswere increased in an IL1b dose-dependent manner (Fig. 3D andSupplementary Fig. S2D). Together, these data demonstrate thatpaclitaxel directs macrophages toward a secretion of mediatorsinfluencing the invasive properties of tumor cells in an IL1b-dependent manner.

IL1b depletion in bone marrow cells or IL1b inhibition usingIL1Ra slightly delays primary tumor regrowth followingpaclitaxel treatment, but increased spontaneous metastases

Having established that paclitaxel administration results in theinduction of host-derived IL1b, which potentially accounts fortumor cell invasive phenotype, we proceeded to investigatewhether depletion of host-derived IL1b before paclitaxel admin-istration affects tumor progression. To this end, we generatedbone-marrow (BM) transplanted WT chimeric mice, in whichBMDCs are either from donor WTmice (WTBMWT) or from donorIL1b�/� mice (WTBM-IL1b�/�

), as indicated in Materials and Meth-ods and as illustrated in Supplementary Fig. S3A. Depletion ofIL1b from BMDCs was confirmed by RT-PCR analysis of periph-eral blood cells (Supplementary Fig. S3B). The chimericmicewereimplanted with LLC-GFPþ cells and when tumors reached 500mm3, themicewere treatedwith paclitaxel and tumor volumewasassessed over time. Treatment efficacy of paclitaxel was slightlygreater in the IL1b-depletedmice (WTBMIL1b�/�

) as compared withtheir WT counterparts (WTBMWT; Fig. 4A). Moreover, depletion ofIL1b from bone marrow cells resulted in a dramatic reduction inthe level of IL1b in tumors ofWTBMIL1b�/�

chimericmice followingpaclitaxel therapy, confirming that inflammatory BMDCs are theprimary source of IL1b in LLC tumor–bearing mice, and nottumor cells (Fig. 4B). Indeed, no significant difference in IL1bexpression was observed between LLC tumor cell lysates from

control mice and mice treated with paclitaxel (SupplementaryFig. S3C). Importantly, histopathological and flow-cytometricanalyses of lungs, revealed a higher number of metastaticlesions in WTBMIL1b�/�mice when compared with WTBMWT

mice (Fig. 4C and D). To test whether increase in metastasiscan be found in an experimental pulmonary metastasis model,indicating whether in IL1b-deprived mice treated with pacli-taxel, tumor cell seeding is altered, LLC cells were intravenouslyinjected to WTBMIL1b�/� or WTBMWT mice that underwent pac-litaxel therapy. No significant differences in mortality rate wereobserved between any of the treated groups, indicating thatIL1b may affect tumor cell dissemination from the primarytumor but not tumor cell seeding (Supplementary Fig. S3D).These results were further reinforced when plasma fromWTBMIL1b�/� mice treated with paclitaxel did not induce LLCand MDA-MB-231 cell invasion when compared with plasmafrom paclitaxel-treated WTBMWT mice (Supplementary Fig.S3E). To rule out compensatory mechanisms that could activateIL1RI, that is, IL1a, in WTBMIL1b�/�mice, anakinra, whichblocks both IL1a and IL1b signaling, was administered incombination with paclitaxel treatment in various tumor-bear-ing mouse cohorts. When administered in a long-term settingstarting one day before chemotherapy administration followedby daily administrations to the experiment's endpoint, ana-kinra slightly but not significantly enhanced the antitumorefficacy of paclitaxel in primary orthotopically implanted4T1 and subcutaneously implanted LLC tumor models, com-parable with previous reported results (ref. 3; Fig. 5A and B).However, in a short-term setting, where anakinra was injectedonly for 3 sequential days starting one day before chemother-apy administration, it did not affect paclitaxel treatment effi-cacy at all (Supplementary Fig. S3F). Moreover, analysis ofhistopathological sections from lungs harvested at the end oflong- and short-term trials, revealed a significantly highermetastatic burden in mice treated with the combination ofpaclitaxel and anakinra or anakinra monotherapy, similarly tothe experiment performed in Figs. 4A and D and 5C and D;Supplementary Fig. S3G. Collectively, these results indicate thatthe long-term blockade of IL1b or its receptor in mice eithertreated with paclitaxel or vehicle control slightly but not sig-nificantly reduces primary tumor growth, but at the same time,it also significantly enhances spontaneous metastases.

Administration of anakinra decreases tumor microvesseldensity yet promotes vessel leakiness

IL1bplays amajor role in tumor angiogenesis, which representsone of the main mechanisms of facilitating tumor invasiveness,and its blockade by IL1Ra was shown to reduce the tumorangiogenic response (17, 18). We therefore further characterizedthe effect of anakinra as a single agent or in combination withpaclitaxel chemotherapy on the tumor's vasculature. CD31 stain-ing of LLC and 4T1 tumors revealed that tumor vessel density wassignificantly lower in tumors treated with anakinra or the com-bination of paclitaxel and anakinra, when anakinra was admin-istered in a long-term setting (Fig. 6A and B). Furthermore, thisreduction in tumor vessel density was accompanied by a higherpercentage of perfused tumor areas as revealed by Hoechst stain-ing (Fig. 6C). Consistently, FITC-conjugated dextran injectionrevealed elevated extravasation of dextran in tumors treated withanakinra or the combination of paclitaxel and anakinra (Supple-mentary Fig. S4A and S4B).

Voloshin et al.





Pro-tumoral (M2-like) tumor-associated macrophages (TAM)are associated with chemoresistance and a higher incidence ofmetastasis formation (12). M2-like TAMsmay also affect vascularleakage and could thus potentially influence tumor perfusion andmetastasis extravasation and dissemination (19). Because proin-flammatory cytokines such as IL1 are abundantly expressed byantitumoral, classically activated (M1-like)macrophages (20), wesought to determine whether treatment with anakinra could altermacrophage polarization toward the M2 phenotype. Flow-cyto-metric analysis of freshly isolated TAMs from 4T1 tumors treatedwith vehicle, paclitaxel, anakinra, or a combination of the twodrugs revealed M1 to M2 phenotype skewing in all treatmentgroups, with significance in the anakinra-treated groups whencompared with control mice (Fig. 6D and Supplementary Fig.S4C). Collectively, these findings may suggest that targeting IL1brestrains primary tumor growth, but also skews TAM polarizationtoward theM2phenotype, which in turnmay account for vascularleakage and tumor cell dissemination, therefore, leading tometas-tasis spread.

DiscussionThis study describes a dual role for chemotherapy-induced IL1b

in tumor progression. Our previous study has shown that pacli-taxel, in addition to its anticancer therapeutic effects, may alsopromote invasive properties in cancer cells via the induction of

host-derived expression of MMP9 in bone marrow cells, thusfacilitating tumor dissemination to distant organs (7). Our cur-rent study demonstrates that in addition toMMP9, paclitaxel alsoinduces the production of IL1b by macrophages, and possiblyother BMDCs (3), thereby promoting tumor cell invasiveness andangiogenesis. We found that the levels of IL1b in the plasmaparalleled MMP9 levels, which could explain the reduced tumorcell invasiveness found in the plasma from mice treated withpaclitaxelþanakinra when compared with plasma from paclitax-el-treated mice. Indeed, Huang and colleagues have recentlyreported that IL1b induces MMP9 expression, leading to meta-static phenotype of gastric adenocarcinoma cells (21). In addi-tion, we found that other tissues, for example, kidney and liverexpressed high levels of IL1b, yet the spleen was the exclusiveorgan tested that exhibited elevated levels of IL1b followingpaclitaxel therapy. Similarly, chemotherapy such as 5-FU andgemcitabine induces IL1b secretion from cells in the spleen, aneffect that is related to the activation of the NOD-like receptorfamily, pyrin domain containing-3 (NLRP3) in the inflamma-some (3). In vitro, paclitaxel induced IL1b production but notsecretion from macrophages at early time points. It is plausiblethat IL1b is secreted at later time points when macrophagesundergo apoptosis following exposure to paclitaxel or whentumor cells exposed to paclitaxel secrete additional stimuli formacrophages to secrete IL1b. It should be noted that IL1b, on itsown, did not result in increased cancer cell invasion phenotype,

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Figure 4.IL1b depletion from bone marrow cells promotes spontaneous metastasis. Half a million LLC-GFPþ cells were implanted to the flanks of chimeric C57Bl/6 micetransplanted with either WT IL1b (WTBM WT) or IL1b�/� (WTBM IL1b�/�) bone marrow cells (n ¼ 6 mice/group). When tumors reached 500 mm3, treatmentwith paclitaxel (PTX) was initiated. A, tumors were measured regularly using Vernier calipers, and tumor volume was assessed over time. B, at endpoint, tumorswere removed and the level of IL1b expression was assessed by ELISA. C, the lungs were removed and evaluated for the percentage of GFPþ cells and thenumber of metastatic lesions. D, representative lung sections (mosaic) and high magnification (zoom factor 3) are provided (scale bar, 2,000 mm). Errorbars, � SD. �� , P < 0.01; �� , P < 0.001.






but only through additional possible mediators secreted by IL1b-activated macrophages. Overall, these in vitro results furthersuggest that additional variables play a role following the induc-tion of macrophage-derived IL1b expression. To further elaborateon this point, we provide several lines of in vivo evidence dem-onstrating the role of IL1b secreted bymacrophages in response topaclitaxel. Specifically, blocking paclitaxel-induced IL1b in mice,either by depleting macrophages or by treatment with a pharma-cologic inhibitor, resulted in a severe reduction in IL1b levels inthe spleen and plasma.Moreover, exposure of different cancer celllines to the plasma of macrophage-depleted, paclitaxel-treatedmice resulted in a drastic decrease in their invasive phenotypewhen comparedwithplasma frompaclitaxel-treatedmice that didnot undergo macrophage depletion. These results are in line withstudies that identified IL1b as an invasion mediating factor, andthat its blockademay therefore prevent prometastatic effects (22).It should be noted that despite the expression of IL1R1 in thecell lines tested, the IL1b-dependent increase in the invasionphenotype of such cells was indirect and could probably resultfrom the pleiotropic effect of IL1b on prometastatic down-stream genes, for example, MMP9 or other proteolytic enzymes,expressed by different host cells, including macrophages(2, 23). We further demonstrated in vivo, that mice that under-went paclitaxel therapy, the depletion of IL1b from BMDCs orthe blockade of its pathway using anakinra, paradoxicallyresulted in increased spontaneous metastasis and poor survivalin both orthotopically and subcutaneously implanted tumormodels. Consistent with our findings, Carmi and colleagues,

have already reported that in IL1b-deprived mice, increasedmetastasis lesions were observed in the lungs of mice bearingan experimental lung metastasis model (24). Taken together,our results suggest that the depletion of IL1b expressed specif-ically by BMDCs induces tumor cell invasion phenotype andtherefore may account for increased metastasis spread.

In the tumor milieu, chemotherapy such as paclitaxel inducesthe secretion of chemotactic factors, such as colony stimulatingfactor-1, which then enhance the infiltration of macrophages andT cells to the treated tumor site, and further promote resistance totherapy (25). TAMs have also been shown to promote tumorgrowth and metastasis by the secretion of paracrine factors,including IL1b as we describe here, that promote angiogenesisand tumor cell invasion and migration (12). Furthermore, theincreased infiltration of macrophages and MDSCs to paclitaxel-treated tumors has been recently shown to induce resistance oftumor cells to therapy, in part due to the secretion of Cathepsin-Bfrom chemotherapy-activated TAMs (2, 3). Therefore, blockingmonocyte and TAM infiltration to treated tumors, or inhibitingtheir activity improves tumor cell chemosensitivity and reducesvascular density (25). However, as shown here, the blockade ofIL1b in the tumor microenvironment polarized TAMs away fromthe M1-like phenotype toward the prometastatic M2-like pheno-type. TAM polarization toward the M2-like phenotype was pre-viously linked to vessel abnormalization (19). Indeed, althoughadditionof anakinra topaclitaxel decreasedmicrovessel density intreated tumors, vessel leakiness was profound, and could explain,albeit indirectly, the increased spontaneous metastasis seen. It is

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Figure 5.Accelerated metastasis spread in tumor-bearing mice treated with paclitaxel and anakinra. A, LLC and (B) 4T1 tumor–bearing mice (n ¼ 7 mice/group) weretreated with paclitaxel, anakinra, or paclitaxelþanakinra as described in Materials and Methods. Tumors were measured regularly using Vernier calipers,and tumor volume was calculated. Tumor growth curves were not statistically different (C and D). At endpoint, lungs were removed for the evaluationand quantification of metastatic lesions in lung sections using H&E staining. Graphs are presented (A and B, relatively to C and D). Error bars, � SD. � , P < 0.05;�� , P < 0.001.

Voloshin et al.





important to note that long-term disruption of the IL1-IL1Rpathway regardless of paclitaxel therapy, promoted M1 to M2TAMphenotype skewing, leading to increased vessel leakiness andsubsequent metastasis spread. Taken together, our data suggestthat IL1b may have dual effect, as an indirect enhancer of theinvasive potential ofmalignant cells, and as an antitumor, inflam-matory-modulator of TAMs, in the tumor milieu.

Another noteworthy preclinical value that can be drawn fromthis study is related to the therapeutic evaluation in preclinicalmodels of add-on drugs to chemotherapy. Bruchard and collea-gues have clearly shown that IL1b blockade increased chemother-apy efficacy, when focusing on its therapeutic activity in primarytumors (3, 26). In ourmodels, we found quite comparable resultswhen treating breast or lung carcinomas with paclitaxel in com-bination with anakinra, although the treatment efficacy of thecombination therapy did not reach statistical significance. How-ever, the combined therapy increased metastatic burden in themice' lungs when compared with mice treated with paclitaxelmonotherapy. When the same therapy was performed on anexperimental lung metastasis model, whereby tumor cells were

injected intravenously via the tail vein and seeded in the lungs, thedepletion of IL1b actually did not significantly change the ther-apeutic activity of paclitaxel. The failure of reproducing therapeu-tic findings of preclinical studies in the clinic is a major concern,and it is related, in part, to the preclinical models used for drugtesting. For example, studies have shown that the combination ofseveral anticancer therapies improve therapy outcome at theprimary tumor level, whereas this treatment is ineffective inmetastatic disease (27, 28). Although anakinra, which presum-ably blocks the IL1 pathway, was theoretically and preclinicallyoffered as an add-on drug to be evaluated in the clinic along withangiogenesis targeting agents (29), recent studies indicated thatanakinra activity might be different between tissues, thereforeallowing diverse and evenopposite effects onhost and tumor cells(30, 31). Indeed, our study provides preclinical evidence for thepossible therapeutic disadvantage of such treatment combina-tions. We demonstrate that the use of long-term IL1b blockadealone or along with chemotherapy, although may slightlyenhance the antitumor activity of primary tumors, it also para-doxically increases spontaneous metastasis. Therefore, such

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Figure 6.Tumors treated with the combination of paclitaxel and anakinra exhibit increased vessel perfusion and M2 macrophage colonization. A, LLC and 4T1 tumor–bearingmice (n ¼ 7 mice/group) were treated with paclitaxel (PTX), anakinra, or paclitaxelþanakinra, in the dose and schedule indicated in Materials and Methods.When tumors reached 500 mm3, they were removed and immunostained with anti-CD31 antibodies (red) to detect endothelial cells, and Hoechst (blue) to detectvessel perfusion (Scale bar, 200 mm). Microvessel density (B) and percentage of perfusion (C) were calculated. D, in parallel, 4T1 tumors were prepared assingle-cell suspensions for the evaluation of M1 and M2 macrophage colonization of tumors using flow cytometry. The number of macrophages was calculated as apercentage per total 106 cells. Error bars, � SD. � , P < 0.05, �� , P < 0.01, �� , P < 0.001.






treatment combinations need to be further evaluated in appro-priate preclinical models. As such, investigating the correlationbetween IL1b and clinical outcome in breast cancer patientstreated with chemotherapy is worthy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T. Voloshin, R.N. Apte, Y. ShakedDevelopment of methodology: T. Voloshin, L. Kaneti, M. Machluf, R.N. ApteAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Voloshin, D. Alishkevitz, V. Miller, E. Isakov,I. Kaplanov, E. Voronov, E. FremderAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Voloshin, D. Alishkevitz, M. Benhar, Y. Shaked

Writing, review, and/or revision of the manuscript: T. Voloshin, R.N. Apte,Y. ShakedStudy supervision: Y. ShakedOther (responsible for the liposome production and analyses): M. Machluf

Grant SupportThis work was supported by grants from the European Research Council

(under FP-7 program, 260633), Israel Science Foundation (490/12), IsraelCancer Research Fund (708/12), and Rappaport funds for Y. Shaked.T. Voloshin was partially supported by the Miriam and Aaron GutwirthStudentship.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 10, 2014; revised March 9, 2015; accepted April 9, 2015;published OnlineFirst April 17, 2015.

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Voloshin et al.




2015;14:1385-1394. Published OnlineFirst April 17, 2015.Mol Cancer Ther Tali Voloshin, Dror Alishekevitz, Limor Kaneti, et al. MetastasisInhibits Primary Tumor Growth but Promotes Spontaneous

Pathway Following Paclitaxel Chemotherapy SlightlyβBlocking IL1

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