Large Molecule Therapeutics

IL6 Receptor Blockade Enhances ChemotherapyEfficacy in Pancreatic Ductal AdenocarcinomaKristen B. Long1, Graham Tooker1, Evan Tooker1, Santiago Lombo Luque1,Jae W. Lee1, Xiaoqing Pan1, and Gregory L. Beatty1,2

Abstract

Inflammation mediated by activation of JAK/STAT signalingis a major cause of chemotherapy resistance in cancer. Westudied the impact of selectively blocking the IL6 receptor(IL6R) as a strategy to inhibit IL6-induced STAT activation andto overcome chemoresistance in pancreatic ductal adenocarci-noma (PDAC). To do this, STAT activation was investigatedin tumors arising spontaneously in LSL-KrasG12D/þ;LSL-Trp53R172H/þ;Pdx-1Cre (KPC) mice. Plasma from patients withPDAC was assessed for its ability to activate STAT3/SOCS3 inhuman monocytes using immunofluorescence microscopy andquantitative gene expression assays. KPC mice and syngeneicmice (wild type and IL6�/�) implanted with KPC-derived celllines were treated with an IL6R-blocking antibody (anti-IL6R).The impact of treatment on tumor growth in KPC mice andmice with KPC-derived tumor implants was monitored using

ultrasonography and calipers, respectively. Tumors were ana-lyzed by IHC to detect changes in STAT activation, tumorviability, and proliferation. We found that STAT3 was the mostactivated STAT protein in PDAC tumors from KPCmice. Plasmafrom patients with advanced PDAC stimulated STAT3/SOCS3activation in human monocytes. In mice, anti-IL6R antibodiestargeted Ly6Chi monocytes, inhibited STAT3 activation intumor cells, and decreased tumor cell proliferation in vivo. IL6Rblockade in combination with chemotherapy induced tumorcell apoptosis, tumor regressions, and improved overall sur-vival. Overall, we show that IL6 signaling drives STAT3 activa-tion in tumor cells and mediates chemoresistance in PDAC.Thus, disrupting IL6 signaling using anti-IL6R antibodiesholds promise for improving chemotherapy efficacy in PDAC.Mol Cancer Ther; 16(9); 1–11. �2017 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDAC) is projected to

become the second leading cause of cancer-related deaths by2030 (1). This dismal outlook associated with PDAC is due atleast in part to its poor responsiveness to standard cytotoxictherapies, including chemotherapy and radiation. A key determi-nant of this treatment resistance is the tumor microenvironment,which demonstrates extensive fibrosis and poor vascularity thattogether result in elevated interstitial fluid pressures capable ofimpeding drug delivery (2–4). In addition, PDAC recruits a robustinflammatory response, composed of immature myeloid cells,macrophages, and granulocytes, which may also limit treatmentefficacy (5–7). Inhibiting the recruitment of myeloid cells totumors using inhibitors of chemokine receptor signaling path-ways (e.g., CCL2/CCR2) can enhance the efficacy of chemother-apy and radiotherapy (8–10). Myeloid cell depletion using a

colony-stimulating factor 1 receptor (CSF1R) inhibitor has alsobeen shown to enhance the efficacy of chemotherapy in PDACmouse models (11). In addition, strategies designed to depleteelements of the extracellular matrix (e.g., collagen and hyalur-onan)havebeen found to improvedrug delivery and the cytotoxiceffects of chemotherapy (2, 3). Tumor-infiltrating myeloid cellscan also be redirected to facilitate depletion of extracellularmatrixcomponents, and in doing so, enhance the efficacy of chemo-therapy (12, 13). Thus, elements of the tumormicroenvironment,including fibrosis and inflammation, are key determinants oftreatment resistance in PDAC.

The JAK/STAT pathway is an important driver of cytokine-mediated cancer inflammation in many human malignancies(14). For PDAC development, activation of the STAT3 pathwayin inflammatory cells is necessary to promote pancreatic intrae-pithelial neoplasia (PanIN) progression (15, 16). Specifically,inhibiting STAT3 activation genetically can block PanIN progres-sion and reduce the development of PDAC in mouse models.However, the development of selective STAT3 inhibitors has beenchallenging, and currently, there are no direct STAT3 inhibitors inclinical trials for cancer.

The importance of the JAK/STAT pathway for defining PDACresistance to chemotherapy has been investigated inmouse mod-els where a nonselective inhibitor of JAK1/2, that blocks signalingvia multiple STAT proteins, including STAT1, STAT3, STAT5, andSTAT6, was shown to improve the activity of chemotherapy (17).Similarly, initial results with the nonselective JAK1/2 inhibitorruxolitinib in combination with chemotherapy (vs. placeboplus chemotherapy) showed early promising activity in a pre-specified subgroup analysis of PDACpatientswith elevated serum

1Division ofHematology-Oncology, Department ofMedicine, PerelmanSchool ofMedicine, University of Pennsylvania, Philadelphia, Pennsylvania. 2AbramsonCancer Center; University of Pennsylvania, Philadelphia, Pennsylvania.

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

CorrespondingAuthor:Gregory L. Beatty, University of Pennsylvania, PerelmanCenter for Advanced Medicine, South Pavilion Rm 8-107, 3400 Civic CenterBlvd., Building 421, Philadelphia, PA 19104-5156. Phone: 215-746-7764. Fax: 215-573-8590; E-mail: gregory.beatty@uphs.upenn.edu

doi: 10.1158/1535-7163.MCT-16-0899

�2017 American Association for Cancer Research.

MolecularCancerTherapeutics

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C-reactive protein (18). However, two randomized phase III trialssubsequently failed to demonstrate clinical benefit with addingruxolitinib to chemotherapy in PDAC patients with metastaticdisease selected on the basis of evidence of systemic inflammation(CRP > 10 mg/dL; ref. 19). Because nonselective inhibition ofthe JAK/STAT pathway can produce significant toxicities, suchas anemia (19, 20) as well as thrombocytopenia, neurotoxicityand increased rates of infection (20), alternative strategies thatselectively inhibit JAK1 activity are being investigated for thetreatment of PDAC, with promising results seen in combina-tion with chemotherapy in a recent early-phase clinical study inmetastatic PDAC (21). Nonetheless, the role of JAK/STATsignaling in determining chemotherapy efficacy remains illdefined.

Here, we examined the effect of targeting the IL6/IL6R signalingpathway as a means of selectively inhibiting STAT3 activation inPDAC for enhancing the efficacy of cytotoxic chemotherapy. Wefound that STAT3 was the dominant STAT protein activatedwithin tumors arising spontaneously in the clinically relevantKrasLSL-G12D/þ, Trp53LSL-R172H/þ, Pdx1-Cre (KPC) mouse model ofPDAC (22). In addition, plasma-derived soluble factors presentwithin the peripheral blood of patientswith advanced PDACwerefound to activate the STAT3/SOCS3 pathway in monocytes dem-onstrating systemic activation of this pathway in patients. Anti-IL6R–blocking antibodies were used to disrupt IL6/STAT3 signal-ing and were found to rapidly bind to a subset of monocytes thatare actively recruited to tumors. Anti-IL6R treatment also inhib-ited STAT3 activation,mainly inmalignant cells, within the tumormicroenvironment, a finding that was reproduced in IL6 knock-out mice, demonstrating the importance of host-derived IL6 inregulating STAT3 signaling inmalignant cells. Finally, therapeuticblockade of IL6R sensitized PDAC tumors to chemotherapy-induced apoptosis, leading to improved survival in tumorimplantationmodels and tumor regressions in KPCmice. Togeth-er, our data demonstrate a role for IL6 in regulating chemosensi-tivity in PDAC. Findings have immediate clinical implicationsgiven the availability of a clinical grade FDA-approved IL6Rantagonist (i.e., tocilizumab) and warrant clinical evaluation ofIL6R-blocking antibodies as ameans for enhancing the efficacy ofcytotoxic chemotherapy in PDAC.

Materials and MethodsAnimals

KrasLSL-G12D/þ, Trp53LSL-R172H/þ, Pdx1-Cre (KPC) mice havebeendescribed previously (22). Thesemice develop PanIN,whichprogresses to invasive PDAC. C57BL/6 (B6) mice (The JacksonLaboratory), IL6-deficient mice (IL6�/�) on the B6 background(The Jackson Laboratory), and normal healthy littermate mice(Trp53LSL-R172H/þ, Pdx1-Cre; PC) on the B6 background were usedin experiments as controls and for tumor implantation studies.Animal protocols were reviewed and approved by the Institute ofAnimal Care and Use Committee of the University of Pennsylva-nia (Philadelphia, PA).

Clinical samplesPlasma was collected by centrifugation of peripheral blood

from patients with newly diagnosed chemotherapy-na€�ve meta-static PDAC. Quantification of IL6 was performed using Luminexbead array technology (Life Technologies) as described previously(23). Written informed consent was required, and the study was

conducted in accordance with the Declaration of Helsinki andapproved by local Institutional Review Boards.

Cell linesPreviously describedmurine PDAC cell lines derived from KPC

mice backcrossed onto the B6 background were used including152.PDA (derived August 2012; ref. 10), 7940B.PDA (derivedJune 2013; ref. 12), and 69.PDA (derived August 2012; ref, 24).Cell lines were authenticated on the basis of histologic analysis ofthe implanted cell line with comparison with the primary tumorfrom which the cell line was derived. Cell lines were negative formycoplasma contamination (tested on October 27, 2016). Celllines were grown in DMEM with 10% FBS supplemented with83 mg/mL gentamicin and L-glutamine.

Animal experimentsKPC mice were monitored weekly for the presence of sponta-

neous pancreatic tumor development. Tumors were detected bypalpation and confirmed by ultrasonography as described previ-ously (13).Micewith tumorsmeasuring approximately 3 to 5mmin diameter were block randomized and enrolled into studies. Fortumor implantation studies, PDAC cell lines were injected sub-cutaneously or orthotopically into syngeneic B6, B6.PC, or IL6�/�

mice as described previously (24). Tumors were allowed todevelop over 14 days to approximately 5 mm in diameter beforebeing enrolled into treatment studies. Tumor volume (mm3) wascalculated asV¼ 1/2 (L�W2) by determining the longest (L) andshortest (W) dimensions using calipers. Mice were treated byintraperitoneal injection of endotoxin-free antibodies purchasedfrom Bio X Cell, including: anti-IL6Ra (clone 15A7) or rat isotypecontrol (clone LTF-2). For survival and tumor growth studies,0.2 mg of anti-IL6Ra or isotype control antibodies were admin-istered twice weekly. Gemcitabine (Gemzar, Eli Lilly) pharma-ceutical grade powder was purchased through the Hospital of theUniversity of Pennsylvania Pharmacy and resuspended in sterilenormal saline at 38 mg/mL 2'-deoxy-2',2'-difluorocytidine. Gem-citabine (120 mg/kg) was administered by intraperitoneal injec-tion on days 0 and 7 of treatment.

Histology, IHC, and immunofluorescence analysisIHC and immunofluorescence staining were performed on

frozen tissue sections. Frozen sections were air dried and fixedwith 3% formaldehyde. Primary antibodies against mouse anti-gens included rabbit anti-pSTAT1 (Cell Signaling Technology),rabbit anti-pSTAT3 (Cell Signaling Technology), rabbit anti-pSTAT5 (Cell Signaling Technology), rabbit anti-pSTAT6(Abcam), rabbit anti-CC3 (Cell Signaling Technology), and ratanti-Ki67 (Dako).

For immunofluorescence staining, sections were blocked with10% normal goat serum in PBS þ 0.1% Tween-20 (PBST). Fordetection of intracellular antigens, following fixation with 3%formaldehyde, tissues were incubated with 100% methanol at�20�C and permeabilized in blocking solution with 0.3% TritonX-100. Tissues were stained with primary antibody in blockingbuffer for 60 minutes at room temperature or overnight at 4�C.Sections were washed in PBST and then incubated with Alexa488-or Alexa568-conjugated goat anti-rabbit or goat anti-rat IgG (LifeTechnologies) for 45minutes at roomtemperature to visualize theantigen of interest. Nuclei were stained with DAPI.

For IHC, endogenous peroxidases were quenched in 0.3%H2O2 inwater for 10minutes and then blockedwith 10%normal

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goat serum in PBST. For detection of intracellular antigens, tissueswere incubated with 100% methanol �20�C and permeabilizedin blocking solutionwith 0.3%Triton X-100. Primary antibody inblocking buffer was applied to tissues for 60 minutes at roomtemperature or overnight at 4�C. Sectionswerewashed in PBS andthen incubated with goat anti-rabbit (Jackson ImmunoResearch)or anti-rat biotinylated IgG (BD Biosciences). Staining wasdetected using VECTASTAIN ABC Kit (Vector Laboratories), andsections were counterstained with hematoxylin.

Brightfield imageswere acquired on aBX43uprightmicroscope(Olympus), and immunofluorescence images were acquired onan IX83 inverted fluorescencemicroscope (Olympus). Immunos-taining was quantified by analysis of 4 to 6 high-power fields pertissue section. Quantification of phosphorylated STAT proteins intissues was determined by calculating the number of cells dem-onstrating pSTAT expression per high-power field.

Flow cytometryPeripheral blood samples were collected from the tail vein of

mice or from human healthy donors. Murine blood samples werecentrifuged and red blood cells lysed using ACK Lysis Buffer(Cambrex/BioWhittaker). PDAC tumors were harvested andminced in digestion media containing collagenase (1 mg/mL),dispase (1 U/mL), and DNase (150 U/mL) and processed asdescribed previously (10). Cell surface molecule analysis ofsingle-cell suspensions obtained from peripheral blood or tumortissue was performed at 4�C for 15minutes in PBS containing 0.2mmol/L EDTA with 2% FCS and analyzed on a FACSCanto (BDBiosciences). Antibodies against mouse antigens were purchasedfrom BD Biosciences unless otherwise specified: CD19 (1D3,APC), CD45 (30-F11, PE-Cy7), F4/80 (eBiosciences, BM8, FITC),Ly6C (AL-21, APC-Cy7), Ly6G (1A8, Percp-Cy5.5), CD3 (BioLe-gend, 1782, Pacific Blue), IL6R (BioLegend, D7715A7, PE), IL6Ra(Bio X Cell, 15A7), biotinylated anti-rat, and streptavidin (PE).Antibodies against human antigens were purchased from BDBiosciences unless otherwise specified: CD56 (NCAM16.2, FITC),CD3 (SK7, PerCP-Cy5.5); CD19 (SJ25C1, PE-Cy7); CD14 (MjP9,APC-Cy7); IL6Ra (BioLegend, 9C4, PE).

ELISA to detect rat antibodiesB6mice received intraperitoneal injectionswith endotoxin-free

antibodies purchased from Bio X Cell, including: anti-IL6Ra(clone 15A7) and rat isotype control (clone LTF-2). Mice weresacrificed at defined time points, and peripheral blood wascollected via cardiac puncture. Blood was spun at 13,000 � g for15 minutes, and serum was collected for analysis. Presence of ratantibodies within the serum was measured using a Rat IgG totalELISA Kit (eBioscience, cat no. 88-50490) according to the man-ufacturer's recommendations.

RNA isolation and qPCRHuman monocytes from healthy donors were preincubated

with or without 0.5 mg/mL tocilizumab and then incubated withor without recombinant human IL6 (0.1 mg/mL), normal donorplasma, or patient plasma for an additional 30minutes in IMDMsupplemented with 1.0% human A/B serum. Tocilizumab(Actemra, Genentech) was purchased through the Hospital ofthe University of Pennsylvania Pharmacy. Following stimulation,culture medium was removed, cells were lysed directly in TRIzol,and RNA was isolated using a Qiagen RNeasy Kit per the man-ufacturer's instructions. cDNA was synthesized from 0.5 to 1.0 mg

of RNA per sample (Applied Biosystems), and primers for qRT-PCR were designed using the Primer 3 online program (25, 26)and synthesized by Integrated DNA Technologies (SOCS3) orpurchased from Applied Biosystems (GAPDH). Relative quanti-fication was measured using either SYBR Green chemistry(Applied Biosystems; SOCS3) or TaqMan chemistry (AppliedBiosystems; GAPDH). SOCS3 expression was normalized toGAPDH, and relative expression was calculated using the DCt

formula. The fold increase or decrease in expression of treatedsamples relative to no treatment controls was calculated (DDCt).Primer sequences for human SOCS3 are as follows: (i) forward: 50-CAAGGACGGAGACTTCGATT-30, (ii) reverse: 50-AACTTGCTGT-GGGTGACCAT-30.

Statistical analysisStatistical analyses were carried out using GraphPad Prism

software. Survival comparison testing was performed using theGehan–Breslow–Wilcoxon test;multiple comparisons testingwasperformed using one-way ANOVA; all other comparisons weredetermined by Student t test or Mann–Whitney test.

ResultsSTAT activation in the tumor microenvironment of invasivePDAC tumors

The JAK/STAT pathway mediates signaling by inflammatorycytokines and is often dysregulated in cancer (14). To investigateJAK/STAT activation in the tumormicroenvironment of PDAC,weexamined the expression of multiple phosphorylated STAT(pSTAT) proteins in invasive PDAC tumors arising spontaneouslyin the KrasLSL-G12D/þ;Trp53LSL-R172H/þ;Pdx1-Cre (KPC) mousemodel. With this approach, we detected high expression ofpSTAT3Tyr705 and pSTAT5Tyr694 in the tumor microenvironmentof late-stage lesions (Fig. 1A and B). We also detectedpSTAT6Tyr641 at low frequency, whereas pSTAT1Tyr701 expressionwas negligible (Fig. 1A and B). We next evaluated the cell typesexpressing pSTAT proteins and based on morphology, localizedpSTAT6Tyr641 expression primarily tomalignant epithelial cells. Incontrast, pSTAT5Tyr694 was seen mainly in stromal cells, andpSTAT3Tyr705 was found in both malignant epithelial cells andinfiltrating stromal cells. We focused our subsequent studies onpSTAT3Tyr705 because it was the most abundantly expressedpSTAT protein detected within PDAC tumors. Using two-colorimmunofluorescence imaging, we localized pSTAT3Tyr705 expres-sion to multiple cell types within the tumor microenvironment,including EpCAMþ malignant epithelial cells, F4/80þ myeloidcells, and aSMAþ

fibroblasts (Fig. 1C). Thus, the predominantSTAT signaling pathways activated within invasive PDAC tissue ofKPC mice were STAT3 and STAT5.

Serum-derived soluble factors from PDAC patients activateSTAT3 signaling in peripheral blood monocytes

Previous studies in the KCmodel of PanIN have implicated IL6as a key mediator of STAT3 activation during PanIN progressionand PDAC development (15). However, the role of IL6/STAT3 inregulating the biology of invasive PDAC remains poorly under-stood, despite its well-recognized association with cachexia,advanced tumor stage, and poor survival (27–29). In our studiesusingKPCmice bearing late-stage tumors in comparisonwith age-matched control littermate mice, we detected increased levels ofIL6 protein in both sera and tumor tissue (Fig. 1D).

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Elevated IL6 levels in the peripheral bloodhavebeenpreviouslyreported to be a potential prognostic factor for chemotherapy-na€�ve patients with PDAC (30). However, increased IL6 levels arenot malignancy specific and can also be seen in benign inflam-matory conditions of the pancreas (e.g., pancreatitis; refs. 31, 32).Consistent with these prior reports, we detected high levels of IL6protein in the plasma of a subset of patients with newly diagnosedmetastatic PDAC (Fig. 2A). IL6 induces STAT3 phosphorylationby binding to IL6Ra and glycoprotein 130 (gp130; ref. 33).Among human peripheral blood mononuclear cells, we foundIL6Ra expression on CD14þ monocytes as well as CD3þ T cells,but not CD19þB cells or CD56þNK cells (Supplementary Figs. S1and S2). Because monocytes are actively recruited to PDACtumors and their infiltration into tumor tissue is associated withtreatment resistance, we hypothesized that plasma-derived solu-ble factors may alter monocyte phenotype and in doing so,regulate treatment resistance in PDAC. Consistent with thishypothesis, we detected increased expression of pSTAT3 inmono-cytes exposed to plasma collected from patients compared withhealthy volunteers (Fig. 2B). We next examined the impact ofpatient versus healthy volunteer plasma on monocyte expressionof SOCS3, a downstream target of STAT3 signaling (33). Here, wealso found that patient (vs. healthy volunteer) plasma induced asignificant increase in SOCS3 expression that was greatest in

patients with high plasma levels of IL6 (>40 pg/mL; Fig. 2B).However, tocilizumab, a human anti-IL6R–blocking antibody,was unable to inhibit increases in SOCS3 expression detected inmonocytes treated with patient plasma (Fig. 2D). Moreover, wefound that IL6, at concentrations detected in the peripheral bloodof patients, was unable to significantly induce STAT3 phosphor-ylation and SOCS3 expression in peripheral blood monocytes(Supplementary Fig. S3). Together, these findings indicate thatplasma-derived soluble factors, present in patients with advancedstage PDAC, can stimulate STAT3/SOCS3 activation in mono-cytes, but this biology is independent of IL6R signaling.

IL6R-blocking antibodies target inflammatory monocytes andenhance the therapeutic efficacy of cytotoxic chemotherapy

STAT3 signaling has recently been shown to be amechanism ofchemoresistance in mouse models of PDAC (17, 34). However,the translation of STAT3 inhibitors to the clinic has been chal-lenging, and thus, this approach is not an immediate therapeuticoption. However, we postulated that inhibition of the IL6/STAT3pathway, bydisrupting the interactionof IL6with IL6Ra,may alsoimprove the sensitivity of PDAC to cytotoxic chemotherapy.Blockade of IL6 signaling using IL6R-blocking antibodies hasbeen used to rapidly reverse symptoms of cytokine release syn-drome (35) and is approved for the treatment of polyarticular

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Figure 1.

IL6 and STAT activation in spontaneously arising murine PDAC. A, Shown are representative images of IHC staining to detect expression of pSTAT1, pSTAT3,pSTAT5, and pSTAT6 in primary pancreatic tumors arising spontaneously in KPC mice. B, Quantification of pSTAT-expressing cells detected by IHC. hpf,high-power field. C, Representative images of 3-color immunofluorescence microscopy to detect pSTAT3 protein expression (green) in EpCAMþ malignantcells, F4/80þ myeloid cells, and aSMAþ myofibroblasts (red). D, IL6 protein levels (pg/mL) detected in serum and pancreatic tissue from tumor-bearing KPC andcontrol littermate mice. � , P < 0.05; �� , P < 0.01; Mann–Whitney test.

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juvenile idiopathic arthritis, rheumatoid arthritis, and systemicjuvenile idiopathic arthritis (36, 37). However, the therapeuticpotential of IL6Rblockade in cancer remains ill defined.We foundin KPC mice and control littermates, as seen in humans, thatIL6Ra was expressed on Ly6Chi F4/80þ inflammatory monocytesaswell asCD3þTcells (Fig.3A;SupplementaryFig. S4).Consistentwith this expression pattern, systemic administration of an IL6Ra-blocking antibody selectively targeted inflammatory monocytesand T cells within the peripheral blood (Fig. 3B). Because inflam-matory monocytes are recognized mediators of chemoresistance,we next examined the binding kinetics of IL6R-blocking antibo-dies to this subset of monocytes in vivo. We administered anti-IL6RamAb (clone 15A7) to mice weighing 20 to 30 g at a dose of

0.2 mg via intraperitoneal injection. This dose is compatible withdoses of human anti-IL6R–blocking antibodies (4–8mg/kg) usedin the treatmentof rheumatologicdiseases (36, 37).We found thatunbound anti-IL6Ra mAb was short-lived within the peripheralblood, lasting only hours after injection compared with isotypecontrol (Fig. 3C). In contrast, we detectedmembrane-bound anti-IL6Ra mAb on inflammatory monocytes for up to 4 days in theperipheral blood (Fig. 3D). On the basis of this pharmacokineticprofile, we adopted a twice-weekly treatment schedule for admin-istration of anti-IL6RamAb to provide continuous IL6R blockadein vivo in mouse models of PDAC.

We next examined the therapeutic impact of administeringanti-IL6RamAbwith orwithout chemotherapy on PDAC growth.

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Figure 2.

Plasma-derived soluble factors from patients with advanced PDAC induce STAT3 activation in human monocytes. A, Shown are protein levels of IL6 (pg/mL)detected in the plasma of patients with newly diagnosed unresectable PDAC in comparison with healthy volunteers. �� , P < 0.01; Mann–Whitney test.B, Representative images of immunofluorescence microscopy showing pSTAT3 expression in monocytes incubated with media alone (Ctrl), media withrecombinant human IL6 (50 ng/mL), or plasma obtained from healthy volunteers (HD) and 2 patients with PDAC with plasma IL6 levels >40 pg/mL. Expression ofSOCS3 in human monocytes incubated with media alone (Ctrl) or plasma obtained from healthy volunteers (HD) and PDAC patients. Low (<40 pg/mL, IL6lo)and high (>40 pg/mL, IL6hi) levels of plasma IL6 were defined on the basis of the median level of IL6 (40 pg/mL) detected in the plasma from 19 patientsexamined. SOCS3 expression was normalized to GAPDH and is shown relative to Ctrl-treated monocytes; n ¼ 3 independent experiments. C, Expression of SOCS3in human monocytes incubated with media alone (Ctrl) or plasma obtained from healthy volunteers (HD) and PDAC patients. Low (<40 pg/mL) and high(>40 pg/mL) levels of plasma IL6 were defined on the basis of the median level of IL6 (40 pg/mL) detected in the plasma from 19 patients examined. SOCS3expression was normalized to GAPDH and is shown relative to Ctrl-treated monocytes; n ¼ 3 independent experiments. D, Shown is SOCS3 expressionnormalized to GAPDH in human monocytes pretreated with or without the IL6Ra-blocking antibody tocilizumab (Toci) and then incubated with media (Ctrl),IL6 (100 ng/mL), or IL6hi plasma from a PDAC patient. � , P < 0.05; ��� , P < 0.001; two-tailed Student t test.

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We first tested this approach in syngeneic B6 mice implantedsubcutaneously with 152.PDA, a cell line derived from a PDACtumor arising spontaneously in KPCmice (10).We found that thecombination of gemcitabine chemotherapy and anti-IL6RamAbdelayed tumor outgrowth and improved overall survival com-pared with either treatment alone (Fig. 4A and B). We then testedthis therapeutic strategy in KPC mice with spontaneously arisingultrasound-confirmed PDAC tumors and detected a significantincrease in tumor regressions measured by ultrasonography at 14days after gemcitabine/anti-IL6RamAb treatment comparedwithbaseline (Fig. 4C). In contrast, we detected no statistically signif-icant effect on tumor growthwith gemcitabine alone. In addition,although a modest slowing of tumor outgrowth in a subset ofmice treated with anti-IL6RamAb alone was observed, this effectwas not statistically significant compared with control-treatedmice.

IL6R blockade inhibits intratumoral Stat3 phosphorylation,decreases tumor cell proliferation, and sensitizes tumors tochemotherapy-induced cell death

We next investigated the mechanism of antitumor activityproduced by treatment with anti-IL6Ra mAb in combination

with chemotherapy. At one day after treatment, we detectedanti-IL6Ra antibodies in the stromal tissue that surrounds malig-nant cells in PDAC tumors (Supplementary Fig. S5). We alsofound that systemic administration of anti-IL6RamAb inhibitedSTAT3 phosphorylation, as seen by IHC, in PDAC tumors arisingin the KPC model (Fig. 5A and B). This decrease in pSTAT3expression was seen mainly in malignant cells. Similarly, wefound a decrease in pSTAT3 expression in malignant cells whenPDAC tumors were grown in syngeneic IL6-deficient (IL6�/�)mice supporting a role for host-derived IL6 (Fig. 5C and D).Although some tumor cells have been reported to express IL6Ra,we found that IL6Ra expression on PDAC tumor cells wasundetectable by flow cytometry (Supplementary Fig. S6). Further-more, IL-6Ra expression was limited to a subset of CD45þ

leukocytes within PDAC tumor tissue (Supplementary Fig. S7).To understand the capacity of anti-IL6Ra mAb to enhance the

sensitivity of tumors to gemcitabine,wenext examined the impactof treatment on tumor proliferation (Ki67) and cell death(cleaved caspase-3). We detected a decrease in Ki67 expressionamong tumors treated with anti-IL6Ra mAb with or withoutgemcitabine chemotherapy compared with control (Fig. 5E andF). However, only the combination of anti-IL6Ra mAb and

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Figure 3.

IL6Ra-blocking antibody 15A7 binds a subset of peripheral blood monocytes in vivo. A, IL6Ra expression on peripheral blood mononuclear cells from controllittermate mice and tumor-bearing KPC mice, n ¼ 3 independent experiments. B, Shown is the percent of peripheral blood cells with surface-boundanti-IL6Ra antibody detected at 10 minutes postinjection; n¼ 8 mice combined from two independent experiments. C, Detection of unbound control or anti-IL6Raantibody in sera at defined time points after injection; n ¼ 3–7 mice per time point. D, Shown is mean fluorescence intensity (MFI) of rat IgG-bound antibodydetected on the cell surface of inflammatory monocytes at defined time points after anti-IL6Ra or isotype control antibody injection in comparison with untreatedmice. n ¼ 3–11 mice per time point for control or anti-IL6Ra antibody–treated mice; � , P < 0.05; �� , P < 0.01; ��� , P < 0.001; two-tailed Student t test.

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gemcitabine produced a significant increase in cleaved caspase-3expression (Fig. 5F; Supplementary Fig. S8). Thus, our findingssupport a role for IL6Ra blockade as a therapeutic approach toinhibit IL6/STAT3 signaling for enhancing the efficacy of cytotoxicchemotherapy in PDAC.

DiscussionThe tumor microenvironment is a major therapeutic barrier in

PDAC. Previous studies have identified poor vascularity anddense fibrosis as physical barriers that may limit the delivery oftherapeutics to the tumor bed (2–4). In this study, we show thatIL6/STAT3 activation is a key determinant of the sensitivity ofPDAC to chemotherapy. Using a clinically relevant genetic mousemodel of PDAC, we found that the tumormicroenvironment wasmarked by preferential and hyperactivation of the STAT3 andSTAT5 signaling pathways. Whereas STAT5 activation was seenwithin stromal cells, STAT3 activation was diffusely detected in

myeloid cells, fibroblasts, and malignant epithelial cells. In addi-tion, soluble factors present in the peripheral blood of PDACpatients induced STAT3 activation and SOCS3 expression inperipheral blood monocytes, indicating systemic activationof the STAT3/SOCS3 pathway. As STAT3 signaling is a majormediator of cancer inflammation and can inhibit the efficacy ofcytotoxic therapies, we hypothesized that IL6-induced STAT3activation would be a therapeutic target for improving chemo-therapy efficacy in PDAC. We found that administration of IL6R-blocking antibodies to inhibit IL6 signaling blocked STAT3 acti-vation in the tumor microenvironment and enhanced the sensi-tivity of malignant cells to cytotoxic chemotherapy. Giventhe availability of clinical-grade IL6R antagonists, our findingshave direct and immediate implications for translating IL6R-blocking antibodies as a strategy to improve the efficacy ofchemotherapy in PDAC.

IL6 has long been associated with advanced tumor stage, poorsurvival, and cachexia in PDAC (27–29). However, to our

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Figure 4.

IL6Ra antibodies enhance the efficacy of gemcitabine chemotherapy in mice with PDAC. B6 mice were implanted with PDAC cells and 2 weeks later treatedas indicated. A, Shown are tumor growth curves (n ¼ 10 mice/group). Statistical significance determined using two-way ANOVA in comparison withcontrol-treated mice. � , P < 0.05; �� , P < 0.01; ��� , P < 0.001. B, Overall survival curves of the four experimental groups (n ¼ 10 mice/group). Ctrl versusanti-IL6Ra, P ¼ 0.0011; gemcitabine (Gem) versus anti-IL6Ra/gemcitabine, P ¼ 0.0042; anti-IL6Ra versus anti-IL6Ra/gemcitabine, P ¼ 0.0425; Gehan–Breslow–

Wilcoxon test. C, KPC mice with ultrasound confirmed tumors were treated weekly with PBS or gemcitabine with or without twice-weekly anti-IL6Ra orcontrol antibody treatment. Shown is a waterfall plot of percent change in primary pancreatic tumor volume at 14 days after treatment relative to baseline with eachbar representing an individual lesion (n ¼ 7–11/group). Anti-IL6Ra/gemcitabine versus Ctrl, P < 0.05, one-way ANOVA.

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knowledge, IL6 has not been previously demonstrated to be atarget for improving cytotoxic chemotherapy in this disease. IL6can activate STAT3 signaling in cells via twomechanisms. The firstinvolves a classical signaling mechanism in which IL6 binds toIL6Ra and gp130 on target cells to induce STAT3 activation.However, only a few cell types, including myeloid cells, hepato-cytes, and T cells, express the membrane-bound form of IL6Ra.This is in contrast to membrane-bound gp130, which is ubiqui-tously expressed by cells. The second mechanism involves IL6binding to a soluble formof IL6Ra (sIL6R) to form a complex thatthen interacts with membrane-bound gp130 to induce signalingin cells lacking IL6Ra, a process termed IL6 trans-signaling. sIL6Rlevels are naturally detected in the circulation (38), but areincreased in patients with cancer that could lead to the formationof IL6/sIL6R complexes and subsequent IL6 trans-signaling(39, 40). Previous work has implicated IL6 trans-signaling as acritical mechanism for STAT3 activation in malignant cells that isnecessary to promote PanIN progression and development ofPDAC (15).

IL6 is a cytokine that has been attributed tomultidrug resistancedue to its ability to modulate the expression of several genesinvolved in regulating survival (e.g., antiapoptotic proteinsincluding Bcl-xL and Mcl-1), proliferation (e.g., Ras/Raf/MEK/MAPK, PI3K/AKT, and JAK/STAT pathways), and cell-cycle pro-gression (41). In PDAC, IL6 has been implicated in the mainte-nance andprogression of pancreatic cancer precursor lesions (42),but its role in defining the biology of invasive PDAC is ill defined.Our findings using IL6R-blocking antibodies show that IL6 isa critical factor for pancreatic cancer cell proliferation in vivo.This finding is consistent with recent studies showing a role forIL6 produced by fibroblasts in supporting pancreatic cancercell growth in tumor organoid models (43). Thus, in the absenceof IL6 as a prosurvival signal, our data suggest that pancreaticcancer cells become more susceptible to chemotherapy-inducedapoptosis.

In our studies, we used anti-IL6Ra antibodies that block IL6binding to IL6Ra and as a result, can be used to inhibit bothclassical and trans-signaling mechanisms of IL6. Using this

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Figure 5.

IL6R antibodies inhibit STAT3 activation, decrease tumor cell proliferation, and sensitize tumors to chemotherapy-induced apoptosis. A, Representativeimages showing IHC staining for pSTAT3 expression in tumor tissue from KPC mice treated with anti-IL6Ra–blocking antibodies or isotype control. Scale bar,100 mm. B, Quantification of pSTAT3þ cells detected in A by IHC. n ¼ 3–4 mice per group, 4 high-power fields per mouse; � , P < 0.05; two-tailed Studentt test. C, Representative images showing IHC staining for pSTAT3 expression in tumor tissue from PDAC cell line implanted orthotopically into syngeneic wild-type(IL6þ/þ) or IL6-deficient (IL6�/�) mice.D,Quantification of the percentage of pSTAT3þ cells per total nucleated cells detected inC by IHC. n¼ 4–5mice per group, 5high-power fields per mouse. ��� , P < 0.001; two-tailed Student t test. E, Tumor-bearing mice were treated with or without anti-IL6Ra for 3 days. Shown arerepresentative images of Ki67 expression in PDAC tumors isolated one day after subsequent treatment with or without gemcitabine chemotherapy. Scale bar,50 mm. F, Quantification of Ki67 and cleaved caspase-3 (CC3) detected in PDAC tumors isolated one day after gemcitabine chemotherapy treatment with orwithout 3-day pretreatment with anti-IL6Ra antibodies. n ¼ 4–5 mice per group, 5–8 high-power fields per mouse. Statistical significance determinedusing two-tailed Student t test. Comparisons were made to Ctrl. �� , P < 0.01.

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strategy, we found that IL6Ra-blocking antibodies enhanced thecytotoxic activity of chemotherapy and inhibited tumor out-growth. However, we were unable to measure significant levelsof IL6Ra expression on malignant cells. As a result, anti-IL6Ra–blocking antibodies may act to inhibit the binding of IL6 tosoluble IL6Ra and in doing so, disrupt IL6 trans-signaling inmalignant cells, a mechanism that has been previously shown tobe critical for the progression of PanIN (15). Consistent with sucha mechanism, we also found that IL6Ra-blocking antibodiescould be detected in the stromal tissue surrounding malignantcell nests. A previous report investigating a nonselective JAK1/2inhibitor for suppressing STAT3 signaling in tumors found thatJAK1/2 inhibition increased microvessel density and enhanceddrug delivery without impacting stromal fibrosis (17). However,we did not observe any significant changes with anti-IL6Ratreatment on vessel patency within tumors. Nonetheless, strate-gies designed to improve macromolecular permeability and drugdelivery (4) could further enhance the therapeutic benefitachieved with IL6Ra blockade. It is also possible, though, thatantitumor activity seen with anti-IL6Ra antibodies used in com-binationwith cytotoxic chemotherapy is dependent ondisruptingIL6 signaling in nonmalignant cells outside or adjacent to tumortissue. Consistent with this hypothesis, IL6 has been shown tostimulate monocyte chemotaxis (44). Thus, IL6 blockade mayregulate monocyte recruitment to tumors or even the phenotypeof tumor-infiltrating monocytes, which we and others have pre-viously shown to be key determinants of the efficacy of cytotoxictherapies, including chemotherapy and radiation (9, 10).

IL6 is a key regulator of STAT3 activation and is detected inPDAC at elevated levels in the tumormicroenvironment aswell asthe peripheral blood. A recent report by Ohlund and colleaguessuggested that the cellular source of IL6 in the tumor microen-vironment of PDAC is mainly nonmalignant cells, includingcancer-associated fibroblasts and tumor-infiltrating leukocytes,

rather thanmalignant epithelial cells (43).Ourfindings using IL6-deficient mice support a role for host-derived IL6 in regulatingSTAT3 activation inmalignant cells. However, even in the absenceof host-derived IL6, we still detected STAT3 phosphorylationin some malignant cells and many nonmalignant cells in PDACtumors. This finding suggests a role for other factors beyondhost-derived IL6 in regulating STAT3 activation in the tumormicroenvironment as well as a role for potentially distinctmechanisms that stimulate STAT3 signaling in malignant andnonmalignant cells.

Tumor-infiltrating monocytes have been found to promotemany of the hallmarks of cancer, including tumor survival,angiogenesis, metastasis, and immune evasion (45). Moreover,the phenotype of circulating monocytes shows inherent plasticityand can be altered even prior to monocyte infiltration into tumortissue (12). We found that soluble factors present within theperipheral blood of patients with advanced PDAC can activateSTAT3/SOCS3 signaling in monocytes in an IL6-independentmanner. Multiple factors may contribute to STAT3/SOCS3 acti-vation besides IL6, including growth factors (e.g., VEGF) andcytokines (e.g., IL10). In our studies, we found that the concen-tration of IL6 detected in the peripheral blood was below thethreshold needed to stimulate STAT3/SOCS3 activation. None-theless, we determined that anti-IL6Ra antibodies bound rapidlyto peripheral blood inflammatory monocytes, which have beenimplicated in chemoresistance (8–10). Although we also foundthat anti-IL6Ra antibodies bound to T cells within the peripheralblood, T cells remained largely excluded from the tumor micro-environment. This finding is consistent with recent work showingthat anti-IL6Ra treatment does not impact T-cell recruitment,even in immunogenic models of PDAC, unless combined withanti-PD-L1 checkpoint therapy (46).

IL6 signaling can be inhibited using either IL6R-blocking anti-bodies or IL6-neutralizing antibodies. IL6R-blocking antibodies

Figure 6.

Conceptual model describing a role forIL6R-blocking antibodies in enhancingthe sensitivity of PDAC tochemotherapy. STAT3 signaling inmalignant and nonmalignant cells in thetumor microenvironment of PDAC isassociated with chemoresistance.Treatment with anti-IL6R–blockingantibodies disrupts IL6/STAT3signaling in both malignant andnonmalignant cells, which suppressestumor proliferation and enhances thesensitivity of PDAC to chemotherapy-induced apoptosis.

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have been mainly used to treat rheumatic diseases, with theexception of cytokine release syndrome induced by chimericantigen receptor T-cell therapy in patients with hematologicmalignancies (35). In contrast, IL6-neutralizing antibodies havebeen studied across a wide range of malignancies, includingmultiple myeloma, renal cell carcinoma, Castleman disease,pancreatic cancer, and prostate cancer (47, 48). As monotherapy,IL6-neutralizing antibodies have not demonstrated significantclinical activity in solid tumors. However, in preclinical modelsof PDAC, IL6-neutralizing antibodies have been shown to stim-ulate T-cell immunosurveillance when used in combination withPD-L1 checkpoint blockade (46). In addition, IL6-neutralizingantibodies have been found to enhance the effects of cytotoxicchemotherapy in several xenograftmodels (49).Our selection of areceptor-blocking antibody versus a cytokine-neutralizing anti-body was based on the poor permeability of the PDAC micro-environment to macromolecules, such as antibodies (4). Despitethis, though, we found that IL6R-blocking antibodies could bedetected in stromal tissue after systemic administration. Thisfinding may reflect active diffusion of IL6R antibodies into thetumor bed. However, it also remains possible that IL6R-blockingantibodies are passively delivered to stromal tissue by binding totumor-infiltrating leukocytes prior to their entry into tumors.Consistentwith this latter possibility, we havepreviously reportedthat an antibody directed against CD40 is delivered to the PDACmicroenvironment by binding to CD40þ tumor-infiltrating mye-loid cells (13). Thus, receptor-blocking versus cytokine-neutral-izing antibodies may act distinctly to alter the therapeutic sensi-tivity of PDAC.

In summary, our findings show that disrupting IL6 signalingusing IL6R-blocking antibodies can shift the tumor microenvi-ronment of PDAC from chemoresistant with hyperactivationof the STAT3 pathway to chemosensitive with decreased STAT3activation (Fig. 6). Thus, we propose that the use of IL6R-blockingantibodies to "condition" tumors and inhibit STAT3 activationis a novel strategy for enhancing the sensitivity of PDAC tochemotherapy.

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

Authors' ContributionsConception and design: K.B. Long, G. Tooker, J.W. Lee, G.L. BeattyDevelopment of methodology: K.B. Long, G. Tooker, S.L. Luque, J.W. Lee,X. Pan, G.L. BeattyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.B. Long, G. Tooker, E. Tooker, S.L. Luque, J.W. Lee,X. Pan, G.L. BeattyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.B. Long, G. Tooker, E. Tooker, J.W. Lee, G.L. BeattyWriting, review, and/or revision of the manuscript: K.B. Long, G. Tooker,J.W. Lee, G.L. BeattyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K.B. Long, S.L. Luque, X. Pan, G.L. BeattyStudy supervision: G.L. Beatty

AcknowledgmentsThe authors thank Michael Kalos and Erica Suppa for human cytokine

analyses, Qian-Chun Yu, Hongwei Yu, and Adam Bedenbaugh for advice andtechnical assistance with IHC assays, and Weijing Sun for helpful discussions.

Grant SupportThis work was supported by NIH grant K08 CA138907 (to G.L. Beatty), NIH

R01 CA197916 (to G.L. Beatty), NIH National Institute of General MedicalSciences K12GM081295 (to K.B. Long), NIH grant F30 CA196106 (to J.W. Lee),a Molecular Biology and Molecular Pathology and Imaging Cores of the PennCenter for Molecular Studies in Digestive and Liver Diseases grant P30DK050306, the Damon Runyon Cancer Research Foundation grant DRR-15-12, forwhichG.L. Beatty is theNadia'sGift Foundation Innovator of theDamonRunyon-Rachleff Innovation Award, by grant number 15-20-25-BEAT from the2015 Pancreatic Cancer Action Network-AACR Career Development Awardsupported by an anonymous foundation, and by grant 2013107 from the DorisDuke Charitable Foundation.

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 December 22, 2016; revised April 17, 2017; accepted May 24, 2017;published OnlineFirst June 13, 2017.

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Published OnlineFirst June 13, 2017; DOI: 10.1158/1535-7163.MCT-16-0899