Malignant Astrocytic Tumor Progression Potentiated by JAK ... · P. Greenﬁeld, New York...

Cancer Therapy: Preclinical

Malignant Astrocytic Tumor ProgressionPotentiated by JAK-mediated Recruitment ofMyeloid CellsPrajwal Rajappa1,William S. Cobb1, Emma Vartanian2, Yujie Huang1, Laura Daly3,Caitlin Hoffman1, Jane Zhang3, Beiyi Shen2, Rachel Yanowitch1, Kunal Garg2,Babacar Cisse1, Sara Haddock4, Jason Huse5, David J. Pisapia6, Timothy A. Chan4,David C. Lyden7,8, Jacqueline F. Bromberg3,8, and Jeffrey P. Greenfield1

Abstract

Purpose: While the tumor microenvironment has beenknown to play an integral role in tumor progression, thefunction of nonresident bone marrow–derived cells (BMDC)remains to be determined in neurologic tumors. Herewe identified the contribution of BMDC recruitment in medi-ating malignant transformation from low- to high-gradegliomas.

Experimental Design: We analyzed human blood and tumorsamples from patients with low- and high-grade gliomas. Aspontaneous platelet-derived growth factor (PDGF) murine gli-oma model (RCAS) was utilized to recapitulate human diseaseprogression. Levels of CD11bþ/GR1þ BMDCs were analyzed atdiscrete stages of tumor progression. Using bone marrow trans-plantation, we determined the unique influence of BMDCs in thetransition from low- to high-grade glioma. The functional role of

these BMDCs was then examined using a JAK 1/2 inhibitor(AZD1480).

Results: CD11bþ myeloid cells were significantly increasedduring tumor progression in peripheral blood and tumors ofglioma patients. Increases in CD11bþ/GR1þ cells were observedin murine peripheral blood, bone marrow, and tumors duringlow-grade to high-grade transformation. Transient blockade ofCD11bþ cell expansion using a JAK 1/2 Inhibitor (AZD1480)impaired mobilization of these cells and was associated with areduction in tumor volume, maintenance of a low-grade tumorphenotype, and prolongation in survival.

Conclusions: We demonstrate that impaired recruitment ofCD11bþ myeloid cells with a JAK1/2 inhibitor inhibits gliomaprogression in vivo and prolongs survival in a murine gliomamodel. Clin Cancer Res; 23(12); 3109–19. �2016 AACR.

IntroductionGlioblastoma multiforme carries a dismal prognosis with a

median survival of approximately 17 months (1). Glioblastoma

multiforme broadly exists as two overlapping entities. In de novoglioblastoma multiforme, the tumor is discovered with a malig-nant phenotype and has pathologic characteristics suggestive of ahigh-grade tumor (1). Secondary glioblastoma multiforme maydevelop as a process of inexorablemalignant transformation froma low-grade phenotype over the course of many years throughmechanisms still being elucidated. Previous studies have sug-gested this process is heavily reliant on the interaction of thetumor with several nontumor cells recruited from nonmalig-nant sites. Relevant components of the tumor microenviron-ment include endothelial cells, fibroblasts, microglia, and bonemarrow–derived myeloid cells (BMDC), which synergisticallypotentiate tumor progression and tumor-associated neoangio-genesis (2–6). The angiogenic switch, which is defined as a stateof rapid tumor growth supported by exponential neovascular-ization during which the malignant phenotype is initiated, is animportant mechanism within low-grade glioma transforma-tion. The distribution of CD11bþ cells within high-gradetumors supports an important role for a myeloid-derived cellpopulation during this process (2–4). Tumor neovasculariza-tion provides nutrients and blood supply to the tumor core andis characterized by a metabolic profile exceeding that of neigh-boring brain parenchyma. BMDCs are key mediators of thisangiogenic switch and initiate, support, and perpetuate malig-nant transformation (2–4).

BMDCs such as macrophages, dendritic cells, neutrophils,eosinophils, mast cells, and myeloid-derived suppressor cells

1Department of Neurological Surgery, Weill Cornell Medical College, New York,New York. 2Weill Medical College of Cornell University, New York, New York.3Department of Medicine, Memorial Sloan Kettering Cancer Center, New York,New York. 4Department of Radiation Oncology, Memorial Sloan KetteringCancer Center, New York, New York. 5Department of Pathology and, HumanOncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center,New York, New York. 6Weill Cornell Medical College, Department of Pathology,Division of Neuropathology, New York, New York. 7Children's Cancer and BloodFoundation Laboratories, Departments of Pediatrics, Cell and DevelopmentalBiology, Weill Cornell Medical College, New York, New York. 8Weill CornellMedical College, New York, New York.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

J.F. Bromberg and J.P. Greenfield contributed equally to this article.

Current address for J. Huse: Department of Pathology and Translational Molec-ular Pathology, University of TexasMDAndersonCancerCenter, Houston, Texas.

Corresponding Authors: Jacqueline F. Bromberg, Memorial Sloan KetteringCancer Center, 1275 York Avenue, Box 397, New York, NY 10065. Phone: 646-888-3112; Fax: 646-888-3200; E-mail: [email protected]; and JeffreyP. Greenfield, New York Presbyterian Hospital, 525 East 68th Street, Whit-ney-605, Box 99, New York, NY 10065. E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-1508

�2016 American Association for Cancer Research.

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(MDSC) are often present in large numbers within the stroma ofneoplasms (7–13). Myeloid cell surface markers include CD11b,CD14, CD34, CD44, CD59, CD68, CD163, and F4/80. MDSCs,another subset of myeloid cells, consist of immature progenitorcells intended for neutrophil, monocyte, or dendritic cell fate.CD11bþ/GR1þ cells are immature myeloid progenitor cells thatcould be classified as MDSCs. CD11b is a marker for myeloidcells from the macrophage lineage and GR1 designates a granu-locytic lineage of origin. CD11bþ/GR1þ cells have been shown inother solid tumors to secrete TGFb, promote angiogenesis, tumorprogression, and metastasis (14).

Both murine and human MDSCs exhibit a CD11bþCD14þ/�

MHCIIlowMHCIþ phenotype; however, those found in mice aredefined as CD11bþGR1þ and can be further divided into twosubtypes: CD11bþGR1hi, which exudes an immature neutrophilphenotype, and CD11bþGR1low, which resembles a monocyticphenotype (12). In our murine model, we selected to examineCD11bþ/GR1þ cells as they are significantly increased (in bonemarrow and blood) during low- to high-grade progression and wewere unable to observe an increase in othermyeloid cellmarkers.Wehave not functionally characterized these CD11bþ/GR1þ cells asMDSCs in our study.

Myeloid cells have been the subject of rigorous investigationwithin the context of solid tumorigenesis and have been shown incertain models to depend on the JAK/STAT3 signaling pathway(15–17). We sought to examine the feasibility of regulatingmyeloid cell recruitment using a JAK 1/2 inhibitor (AZD1480)initially developed for the treatment of myeloproliferative dis-orders. AZD1480was shown to restrictmyeloid cell accumulationwithin the tumor microenvironment and impair tumor progres-sion in murine models (18, 19). Here, we demonstrated that thetransition from low- to high-grade glioma was associated withincreased BMDCs both within the circulation and tumor. Impor-tantly, this process was potently blocked with AZD1480 treat-ment, leading to a survival advantage in murine models. Theseresults suggest a novel therapeutic approach for the managementof low-grade gliomas and inhibition of malignant progression.

Materials and MethodsCell lines

A Ntv-a RCAS high-grade cell line was generated through thehomogenization of high-grade tumors derived from explantsharvested from 10-week-old Ntv-a tumor bearing mice. Tumortissues were minced and subsequently incubated at 37�C withPAPAIN (0.1 mg/mL) in PIPES buffer with DNAase. Tissuesuspensions were then gently shaken for 20 minutes. Afterquenching the digestion with FBS, the suspension was againdisruptedwith apipette andpassed through a100-mmcell strainer(BD Biosciences). Cells were collected by centrifugation (1,300rpm � 5 minutes) and seeded onto poly-L-lysine–coated petri-dishes with complete medium containing 10% FBS in DMEMovernight. Cell lines were subsequently grown and passaged incomplete medium. The cell line described above is not authen-ticated. Adherent glioblastoma multiforme–derived cell lines(U87 and U251) and oligodendroglioma-derived neurosphereshave been described previously (20, 21).

Drug preparation and administrationAZD1480 was purchased from ChemieTek. For in vitro experi-

ments, AZD1480 was dissolved in 1% DMSO. For in vivo experi-ments, AZD1480 was dissolved in water, 0.5% hypermethylcel-lulose (HPMC), and 0.1% Tween 80. At 3 weeks of age, animalsbegan treatment with AZD1480, which was administered via oralgavage once a day, 5 days a week, over 3 consecutive weeks. Thetreatment course was stopped at 6 weeks of age for the short-termgroups, whereas it was continued indefinitely in the long-termcohorts. For transplant cohorts, treatment initiation was started at7 weeks of age and was administered for 3 weeks until animalsreached 10 weeks of age. The drug was administered through anoral gavage needle at a dose of 60mg/kg.Control animals receivedidentical volumes of vehicle without AZD1480 by oral gavage.

Tumor-generating miceThe Ntv-a model system was utilized as described previously.

To derive glioma-bearingmice,Ntv-a (WT)mice weremated withNtv-a Ink4aArf�/� LPTEN mice to produce offspring (22–26).RCAS-hPDGFB-HA and RCAS-Cre vectors were transfected intoDF-1 chicken cells (ATCC) using the FuGENE 6 Transfection Kit(Roche). DF-1 cells transfected with RCAS-PSG were cultured in10% FBS-DMEM under standard conditions. For tumor induc-tion, transfected DF-1 cells were trypsinized, centrifuged, andinjected into the brain parenchyma of Ntv-a mice with a hetero-zygous Ink4a/Arf þ/�, PTENfl/fl background at postnatal day 0–2(24, 25, 27).

Magnetic resonance imagingAll animals were imaged with a Bruker Mouse Brain Surface

Coil (Bruker Biospin MRI, Inc) while anesthetized under 1%–2%isoflurane (Baxter). Images were acquired on a Bruker BioSpec70/30 USR 7T (Bruker Biospin MRI, Inc) utilizing gadoliniumcontrast at a 9:1 saline to Magnevist (Bayer Healthcare Phar-maceuticals Inc) ratio. The mouse brain was imaged in the axialorientation using RARE T1-weighted and TurboRARE T2-weighted fast spin-echo rapid acquisition. T1 sequence repeti-tion time was 1,500 milliseconds with echo time ¼ 17.4milliseconds and an echo train length of 6. T2 repetition timewas 2,014milliseconds with echo time¼ 48.4 milliseconds andecho train length of 10. For both T1 and T2, the field of view

Translational Relevance

Ineffective CNS drug penetration through the blood–brainbarrier for brain tumors has stalled the development of effi-cacious treatment paradigms. Themajority of current first-linetherapies for low-grade and high-grade gliomas rely on inhi-biting tumor cells: our work is innovative in that it directlytargets bone marrow-derived cells (BMDC) within the tumorextrinsic microenvironment. Without having a direct effect onthe CNS, we modulate the tumor microenvironment anddelay progression of disease, which has major clinical rele-vance. Roughly 40% of gliomas present as lower grade lesionsthat progress to more invasive, malignant phenotypes. Theseslow growing, indolent tumors have the capacity to transforminto highly malignant and aggressive high-grade gliomas.Despite standard therapeutic regimens, transformation tohigh-grade glioma is common. These data suggest a potentialopportunity to consider treatment with JAK inhibitors inpatient cohorts that show an upregulation of BMDCs in theirperipheral blood prior to transformation.

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was 20 � 20 mm with slice thickness of 1.0 mm and spatialresolution of 0.078 � 0.078 mm/pixel. Tumor progression wascharacterized between 2 and 12 weeks.

Tumor volumetric analysisT1- or T2-weighted MRI images representing axial sections of

subject brains were uploaded into ImageJ software. The slicehalfway through each animal's dataset, typically image 7 of 14,was determined to show the most representative image oftumor development and size. These slices were subsequentlyanalyzed using the Free Hand Tracing and Measure tools tooutline the two-dimensional area of brain tissue present. Traceswere initiated slightly posterior to the ventral aspect of the skulland followed dorsally, forming a closed loop with the initialtrace point. The area formed by this outline was then calculatedin square millimeters using the Measure function. Each pixelrepresents 1 mm of depth; thus, overall brain volume wasobtained by multiplying measured area by 1 mm. Animal brainvolumes for controls and experimental conditions were aver-aged per group and presented graphically. In an attempt tominimize baseline differences in brain size and development,comparative volumetric analysis was only performed betweenanimals treated under the same experimental conditions andfrom the same litter.

Brain tissue processingAnimals used for histologic examination were sacrificed using

carbon dioxide (CO2) and brains were removed and fixed in4% paraformaldehyde for 24 hours followed by dehydration in70% ethanol. The fixed, paraffin-embedded brain tissues wereserially sectioned (10 mm), slide-mounted, and then deparaffi-nized in Histoclear (Allan Scientific) and soaked in serial alcoholgradients.

Flow cytometry analysisFlow cytometry analysis was performed as described previ-

ously (19). Peripheral blood drawn from human subjects withhistologically confirmed grade II (n ¼ 10) and IV (n ¼ 10)gliomas and healthy controls was centrifuged at 500 � g for 10minutes at 4�C. Isolation of white blood cells was performedusing a Ficoll-Paque Gradient (GE Healthcare) and stained withCD11b antibody (BD Biosciences 1:50). Blood collected fromnontumor controls was used solely for the purpose of com-parison between low- and high-grade counterparts for circu-lating CD11bþ cells. Blood and bone marrow from mice wereharvested and stained with CD11b (BD Biosciences 1:200) andGR1 antibodies (BD Biosciences 1:100).

Adoptive bone marrow transplantationTransplants were performed with GFPþ bone marrow from

age-matched donor Black 6 (BL6) mice. Bone marrow washarvested by flushing the femurs of BL6/GFPþ donor mice. Atotal of 5 � 106 bone marrow cells from BL6/GFPþ donor micewere transplanted into lethally irradiated (9.5 Gy) 2-week-oldNtv-a heterozygous knockout mice by retro-orbital injection.During the irradiation procedure, mice heads were shieldedwith a custom-designed lead helmet apparatus. After 4 weeks,GFPþ bone marrow–reconstituted animals (day 49) werestarted on a 3-week treatment regimen as previously described.Animals were sacrificed at week 11 (day 77). Brain tissues were

dissected and fixed in 4% paraformaldehyde or in a mix of 2%paraformaldehyde and 20% sucrose solution overnight andembedded in Tissue-Tek optimal cutting temperature medium(OCT; Electron Microscopy Sciences). Once embedding medi-um was in place, the OCT blocks were frozen over dry ice. Theseblocks were sectioned at 10-mm thickness using a cryotome(Leica) and analyzed for GFPþ fluorescence and subsequentimmunostaining (CD11b, pSTAT3).

Cell proliferation and Western blot assaysAn RCAS high-grade cell line was cultured at a density of 105

cells per well in 96-well plates (Corning). Using the RCAS high-grade cell line, AZD1480 was introduced at 1.0 mmol/L con-centration and cells were counted after coincubation for both48 and 72 hours. U87 and U251 cells were cultured in completemedium, and TS603 cells were cultured in Neurocult baselmedium (StemCell Technologies) containing Neurocult sup-plement, EGF, FGF, and Heparin. To measure cell growth,3–5 � 104 cells were seeded per well with either AZD1480(0.5 mmol/L and 1 mmol/L) or DMSO. Cells were then countedat 24-, 48-, and 72-hour time intervals. Each experiment wasperformed in triplicate and cell counting was performed using ahemocytometer.

Western blot analysis was performed as described previously(19). All of the glioma cell lines were treated with AZD1480 for 5hours before cell lysis for the analysis of pSTAT3 (Y705) and totalSTAT3 (Cell Signaling Technology).

ImmunohistochemistryIHC was performed on a Leica Bond system using the

standard protocol. For IDH1 (R132H) staining, tissue sectionswere subjected to antigen retrieval using a Tris-EDTA buffer for20 minutes and incubated with DIA-H09 (1:50) for 15 minutesat room temperature. For CD11b staining, the sections weresubjected to antigen retrieval using a sodium citrate buffer for30 minutes and incubated with anti-CD11b (ab52478 1:100)for 25 minutes at room temperature. Signals were detectedusing an HRP-conjugated compact polymer system and visu-alized using DAB as the chromogen. Each tumor was scored aspositive if at least one tumor core showed >10 CD11bþ cellswith circumferential staining.

ImmunofluorescenceTo assess the degree of pSTAT3 staining in human gliomas,

two separate tissue microarrays comprising formalin-fixed,paraffin embedded (FFPE) tissue cores of human low-and high-grade gliomas were stained with pSTAT3 (Tyr705;0.2 mg/mL; Cell Signaling Technology) and counterstainedwith DAPI. Immunofluorescence experiments were performedusing Discovery XT processor (Ventana Medical Systems) at theMolecular Cytology Core Facility (MSKCC, New York, NY). Theslides were scanned at high resolution and analyzed usingPanoramic Viewer Software. Up to 3 tissue cores per case werescanned at low power for an area of maximal pSTAT3 staining.A representative 400� field from this area was selected forquantification. Distinct nuclear staining above background wasconsidered positive. In each image, 100 to 500 cells weremanually counted using ImageJ software's cell counter andgrid macros. A two-tailed Student t test was used to assess for asignificant difference in positive pSTAT3 staining between

JAK Inhibition Impairs Glioma Progression

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high-grade glioma (n ¼ 41) and low-grade glioma (n ¼ 38)samples (total n ¼ 79). Multiplex immunofluorescent stain-ing for pSTAT3/GFP/CD11b was performed as describedpreviously (28).

Statistical analysis and quantificationData is represented as mean � SD. N values indicate the

number of animals or slides used for interpretation of data fora particular experimental animal cohort. Tumor histology wasanalyzed in a blinded fashion for experimental cohorts andquantified for hallmark characteristics of gliomas by a neuropa-thologist. Significant differences between experimental groupswere measured by a Student t test with P values indicated lessthan 0.05 (�) or 0.01 (��).

ResultsCD11bþ myeloid-derived cells are increased in high- versuslow-grade gliomas

We hypothesized that CD11bþ BMDCs contribute to thetransition of low- to high-grade gliomagenesis. To test thishypothesis, we collected peripheral blood from patients withlow-grade gliomas, high-grade gliomas, and non-tumor controls.Flow cytometry was used to measure CD11bþ cells, demonstrat-ing an increased number of circulating CD11bþ cells in patientswith high-grade gliomas (66%; grade IV) as compared with low-grade gliomas (42%; grade II; Fig. 1A). Next, we examinedwhether these findings could be recapitulated in a PDGF-drivenmurine model of low- to high-grade glioma progression(Fig. 1B). This animal model, termed Replication-Competent

Figure 1.

Elevated CD11bþ expression in myeloid cells is associated with malignant human and murine gliomas. A, Flow cytometric analysis of CD11bþ cells from theperipheral blood of patients with low (n ¼ 10) and high grade (n ¼ 10) glioma (WHO grade II and IV; n ¼ 20), ��P ¼ 0.01. B, RCAS murine modeldepicting low to high-grade transition (3–12 weeks) and representative tumor histology at each stage. C, Flow cytometric analysis of CD11bþ/GR1þ cellsin blood of RCAS mice bearing low- (n ¼ 7) and high-grade tumors (n ¼ 9). Quantification of CD11bþ/GR1þ cells at normal (n ¼ 4), low- (n ¼ 7), andhigh-grade tumor (n ¼ 9) stages, Student t test, �P < 0.05. D, Flow cytometry analysis of CD11bþ/GR1þ cells in bone marrow from RCAS mice bearing low-(n ¼ 7) and high-grade tumors (n ¼ 9). Quantification of percentage of CD11bþ/GR1þ cells at normal (n ¼ 4), low- (n ¼ 7) and high-grade (n ¼ 9)tumor stages is shown on the right. Student t test, �P < 0.05.

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ASLV long terminal repeat (RCAS), allows for a murine retro-virus (MoMuLV) to deliver the PDGF-B oncogene into glialprogenitor cells of mice deficient for the tumor suppressor PTENand heterozygous for Ink4a/Arf (Nestin-tva, P16Ink4aþ/�P14Arfþ/

�PTENfl/fl; Fig. 1B; Supplementary Fig. S1A; refs. 24, 25, 27).Following tumor induction, mice first developed low-grade gli-omas (3–4 weeks), which transformed into high-grade gliomas(7–10 weeks; Fig. 1B). We examined the number of CD11bþ/GR1þ cells in the peripheral blood (Fig. 1C) and bone marrow(Fig. 1D) of non-tumor bearing mice, low-grade gliomas, andhigh-grade gliomas by flow cytometry.We observed an increase ofCD11bþ/GR1þ cells in the blood and bone marrow of micebearing high-grade gliomas as compared with low-grade andnon-tumor controls.

We thenhypothesized that these circulatingmyeloid cells couldinfiltrate the tumor microenvironment during the low- to high-grade transformation. Accordingly, we examined CD11bþ cells inhuman and murine low- and high-grade gliomas by immuno-histochemical (IHC) and immunofluorescent (IF) analyses.Increased CD11bþ staining was observed in high-grade gliomasas compared with low-grade gliomas (Supplementary Fig. S1B).

In addition, consistent with the literature, increased vascularitywasobservedbyCD31þ staining in thehigh-grade tumors (ref. 29;Supplementary Fig. S1B).

These data demonstrate that the process of glioma transi-tioning from a low- to high-grade state is associated with anincreased number of CD11bþ cells in the peripheral circulationand within the tumor of high-grade gliomas.

BMDCs are recruited to the tumor microenvironmentThe above data led us to hypothesize that as low-grade gliomas

transition to a high-grade state, CD11bþ cells are mobilized fromthebonemarrow into the peripheral circulation and subsequentlyare recruited to the tumormicroenvironment. To test this hypoth-esis and differentiate between recruited and resident CD11bþ

cells, we transplanted GFPþ bone marrow into the PDGF murinegliomamodel (GFPnegative; Supplementary Fig. S2).We comparedthe number ofGFPþ/BMDCsþ in the low- andhigh-grade gliomasby IF and flow cytometry. A (30-fold) increase in the number ofGFPþ/BMDCsþ was observed in high- (week 8) versus low-gradegliomas (week 4; Fig. 2A and B). In addition, the GFPþ/BMDCsþ

were found in a perivascular distribution (Fig. 2A).

Figure 2.

Bone marrow–derived cells (BMDC) are recruited to the tumor microenvironment. A, Representative photomicrographs of tumor cross sections from GFPþ bonemarrow–transplantedmice counterstainedwith CD31þ at low- (n¼4) and high-grade stages (n¼4; 4 and 8weeks, respectively). Scale bar, 50mm.B,Representativeflow cytometric analysis of brain tumor tissue from RCAS murine animals gated for GFPþ cells in low- (n ¼ 4) and high-grade tumors (n ¼ 4), �P < 0.05. C,Confocal microscopic analysis of high-grade tumor sections (n¼ 3 animals, >6 sections/tumor) stained for DAPI and CD11b from GFPþ bone marrow–transplantedRCAS mice. Scale bar, 50 mm. D, Quantification of cells labeled with GFPþ, CD11bþ, and costained (GFPþ/CD11bþ). Percentage of CD11bþ/GFPþ cells.






To determine what proportion of these GFPþ/BMDCsþ weremyeloid-derived, we performed a GFPþ/CD11bþ costain oftumor sections. Of the GFPþ/BMDCsþ recruited population,approximately 33% of these cells were CD11bþ (Fig. 2C andD). These data demonstrate that a third of recruited tumor-infiltrating myeloid cells (CD11bþ) are derived from the bonemarrow.

JAK 1/2 inhibition following bone marrow transplantationmitigates GFPþ/BMDC recruitment and tumor burden

The IL6/JAK/STAT3 signaling pathway has been shown topromote tumorigenesis in part through the expansion and activityof CD11bþ myeloid cells in models of renal and breast cancer(19, 30). We hypothesized that preventing the expansion andmobilization of these cells with an inhibitor of this pathway couldabrogate the transition from low- to high-grade glioma. To testthis hypothesis, we utilized the transplanted GFPþ bone marrowin the PDGF murine glioma model (Fig. 3A; Supplementary Fig.S2). Upon radiographic evidence of low-grade tumors, animalswere treated with a JAK 1/2 inhibitor (AZD1480) for 3 weeks (Fig.3A). ByMRI, vehicle control–treated animals had increased tumorburden as compared with AZD1480-treated animals (Fig. 3B). Todetermine whether tumors fromAZD1480-treatedmice harboredfewer BMDCs as compared with vehicle control–treated mice,animals were sacrificed and tumors were analyzed for GFPþ cellrecruitment within brain tumors. Vehicle control–treated animalsdemonstrated a (10-fold) increase of GFPþ cells within the tumoras compared with AZD1480-treated animals (Fig. 3C). Theseresults demonstrate that AZD1480 blocks the recruitment ofBMDCs to the tumor, which correlates with a reduction intumor burden. Also, to further examine the effect of AZD1480on BMDCs and the resident microglial population, we per-formed a costain with GFPþ/CD11bþ on these tumors. Vehiclecontrol–treated animals demonstrated a (10-fold) increase inGFPþ/CD11bþ cells within the tumor as compared withAZD1480-treated animals (Fig. 3D). In addition, vehicle con-trol animals demonstrated a (5-fold) increase of residentmicroglia (CD11bþ/GFP�) when compared with AZD1480-treated animals (Fig. 3D). Coincident with a reduction ininfiltrating myeloid cells (CD11bþ/GFPþ) we also observed a(5-fold) reduction in CD11bþ/pSTAT3 cells in AZD1480–trea-ted animals when compared with vehicle control animals(Supplementary Fig. S3C and S3D). These data suggest thatAZD1480 blocks the recruitment of CD11bþ cells to the tumor,and decreases the number of resident microglia.

JAK 1/2 inhibition prevents low- to high-grade tumorprogression

These data led us to hypothesize that AZD1480 impairs thetransformation from low- to high-grade glioma in part througha blockade of BMDC recruitment. Mice with radiographicallyconfirmed low-grade gliomas were treated with AZD1480. Incontrast to the cohort described above, nontransplanted micewere used, as radiation (required for bone marrow transplant)delays tumor formation (31). Mice with radiographically con-firmed low-grade tumors were treated for 3 weeks with eitherAZD1480 or vehicle control (Fig. 4A). We assessed tumorburden in these animals by MRI and performed hematoxylinand eosin (H&E) staining of the brain sections. MRI revealedreduced tumor burden in AZD1480-treated animals whencompared with vehicle control–treated animals (Fig. 4B).

Morphologic examination of tumors from vehicle control ani-mals by H&E confirmed features consistent with glioblastomamultiforme including increased cellularity, pseudopalisadingcytoarchitecture, nuclear pleomorphism, hemorrhage, andnecrosis, along with microvascular proliferation and mitoticfigures (Fig. 4B; Supplementary Fig. S3). In contrast, theAZD1480-treated animal brain tumors had decreased micro-vascular proliferation and no mitotic figures present andappeared less aggressive morphologically. (Fig. 4B; Supplemen-tary Fig. S3A) To examine the effect of AZD1480 on theproduction and mobilization of CD11bþ/GR1þ cells, flowcytometry was performed on the bone marrow and blood ofanimals treated with vehicle control and AZD1480. After 3weeks of treatment with AZD1480, animals showed a 7-foldreduction in CD11bþ/GR1þ cells in the peripheral blood whilea 2.5-fold reduction of these cells was observed in the bonemarrow (Fig. 4C). These data demonstrate that AZD1480impairs tumor progression and inhibits the mobilization ofCD11bþ/GR1þ cells. Given these observations, we then askedwhether transient or short-term treatment regimen would havean impact on survival while minimizing potential systemictoxicity. The transient or short-term treatment approach inclu-ded a 3-week regimen with AZD1480 until 6 weeks of age. Oncelow-grade tumor formation was confirmed radiographicallyaround 3 weeks of age, animals were treated with AZD1480or vehicle control for a duration of 3 weeks. Animals wereobserved for clinical symptoms of progression (macrocephaly,weight loss, hunched back, poor feeding, and/or immobility;ref. 27). The survival of AZD1480-treated animals when com-pared with vehicle control–treated animals was increased byapproximately 2-fold (Fig. 4D). A long-term or continuoustreatment approach was also utilized to assess overall survivalwhere animals were treated with AZD1480 or vehicle controlstarting at 3 weeks indefinitely until death. Similarly, thesurvival of AZD1480-treated animals when compared withvehicle control–treated animals was increased by (2-fold) withan indefinite treatment regimen (Fig. 4E).

In addition to increased vascularity and increased recruitmentof CD11bþ cells in high-grade gliomas, we also examined pSTAT3levels (a JAK 1/2 target) by IHC in human high- and low-gradegliomas. As expected, human high-grade gliomas had increasedpSTAT3 staining when compared with low-grade gliomas (ref. 32;Supplementary Fig. S4A). Given the paucity of pSTAT3 in low-grade tumors, our data suggest that AZD1480 prevents low- tohigh-grade transformation by impairing bone marrow–derivedcell recruitment.While the presence of pSTAT3 in tumor cells doesnot necessarily predict a dependence on pSTAT3 for growth, ourdata suggest that AZD1480 had an effect on the low-grade tumormicroenvironment by impairing the mobilization of BMDCs.Similar to human low- and high-grade gliomas, we performedpSTAT3 IHC on vehicle control and AZD1480-treated animalbrain sections which demonstrated increased pSTAT3 staining invehicle control–treated animals when compared with AZD1480-treated animals (Supplementary Fig. S4B).

PSTAT3 has been shown in previous studies to be highlyexpressed in tumor cells along with microenvironment cellsincluding BMDCs (15, 19, 33). We treated a small cohort (n ¼5) of animals with radiographically confirmed high-grade glio-mas for 1 week with AZD1480 and no impact on survival wasobserved (the mice all died within one week). In addition toAZD1480's direct effect on targeting pSTAT3 in BMDCs, we

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Figure 3.

JAK 1/2 inhibition following bone marrow transplantation mitigates CD11bþ/GR1þ-mediated low-grade glioma transformation. A, Schematic of RCASlow-grade murine glioma bearing animals postirradiation (2 weeks) and GFPþ bone marrow transplantation, treated with AZD1480. B, Representativeimage of T2-weighted 7T MRI of vehicle control (n ¼ 7) and AZD1480-treated (n ¼ 7) animals at 10 weeks. Animals were imaged around 7 weeks. C,Representative photomicrographs of tumor cross sections from mice treated with vehicle control (n ¼ 5) and AZD1480 (n ¼ 5). Quantification of averagenumber of GFP cells per high power field in bar graph format, (n ¼ 10). Scale bar, 50 mm, ��P ¼ 0.004. D, Representative photomicrographs of tumorcross sections from mice treated with vehicle control (n ¼ 5) and AZD1480 (n ¼ 5) costained with GFPþ and CD11bþ. Quantification of average number of cellsper high power field in bar graph format labeled with GFPþ, CD11bþ and double labeled with GFPþ/CD11bþ, (n ¼ 10). Scale bar, 50 mm, �P < 0.05.






examined its effects on high-grade tumor cell proliferation in vitro(Supplementary Fig. S4C). AZD1480 at 1.0 mmol/L (which effec-tively blocked pSTAT3 expression) had no effect on cellularproliferation when compared with vehicle control–treated cells(Supplementary Fig. S4C and S4D). In addition to AZD14800sdirect effect on targeting pSTAT3 in BMDCs, we examined its

effects on murine high-grade and human-derived glioma cellproliferation (TS603, U87, U251; Supplementary Fig. S4C andS4D). AZD1480 at 0.5 and 1.0 mmol/L (which effectively blockedpSTAT3 expression) had no effect on cellular proliferation whencompared with vehicle control–treated cells (Supplementary Fig.S4C and S4D).

Figure 4.

JAK 1/2 inhibition prevents low- to high-grade tumor progression. A, Animal schematic depicting experimental design to examine effect ofAZD1480 (treatment for 3 weeks) on tumor progression. Treatment with AZD1480 was stopped at 6 weeks. B, Representative images of T2-weighted7T MRI of vehicle control (n ¼ 9) and AZD1480 (n ¼ 9) treated animals at 6 weeks. Representative images of H&E staining of tumor sections fromcontrol (n ¼ 8) and AZD1480 (n ¼ 8) treated animals at 6 weeks. Scale bar, 100 mm. C, Representative flow cytometry graphs of CD11bþ/GR1þ fromblood (n ¼ 3/group) and bone marrow (n ¼ 7/group) of vehicle control and AZD1480-treated tumor bearing mice at 6 weeks. �P ¼ 0.0386 blood;P ¼ 0.0556 bone marrow (BM). D, Kaplan–Meier symptom-free survival curve for RCAS mice treated for 3 weeks only with vehicle control (n ¼ 9)and AZD1480 (n ¼ 9), P < 0.0001. E, Kaplan–Meier symptom-free survival curve for RCAS mice treated indefinitely with vehicle control (n ¼ 6)and AZD1480 (n ¼ 6), P < 0.0001.

Rajappa et al.





DiscussionThe role of BMDCs in promoting angiogenesis and invasion

during tumor progression has been previously described in cancer(7, 8). However, the multiple molecular mechanisms convergingwithin the microenvironment to promote tumor progression inlow-grade gliomas remain under investigation. Ultimately, therole of BMDCswithin themicroenvironment during low- to high-grade glioma transformation in this patient population is poorlyunderstood. There exists a delicate balance that is achievedbetween the tumor and stroma; we suggest that one of the earliestdemarcations of malignant progression in gliomas is the expan-sion of the tumor-associated stroma including a large infiltrationof BMDCs.

Recent studies have described the importance of CD11bþ/GR1þ cell recruitment and infiltration in solid tumors (9, 10,14, 29, 34). These studies demonstrate that myeloid cells supportthe tumor endothelium by producing proangiogenic elementssuch as matrix metalloproteinase 9 (MMP-9; refs. 15, 18),highlighting a role formyeloid cell–mediated angiogenesiswithinthe microenvironment. The STAT3 signaling pathway has beenshown to be critical for myeloid-cell dependent tumor angiogen-esis within multiple murine models including B16 melanoma,4T1, andpolyomamiddle tmurine breast cancermodels (19, 33).We explored a similarly predicted role for CD11bþ/GR1þ cellsmediating tumor progression within the glioma microenviron-ment and via JAK/STAT signaling with our PDGF-driven RCASmurine glioma model. This model lacks IDH alterations oftenseen in amajority of diffuse astrocytomas (35, 36); however, giventhat we are not studying the molecular pathways of gliomagen-esis, rather the tumor microenvironment–immune system mod-ulation, we believe this is a usefulmodel given its wide utilizationand our internal controls using other nonspontaneous mousemodels (20).

We have studied the modulation of the glioma microenviron-ment through BMDC recruitment during transformative glioma-genesis and the influence of JAK/STAT signaling. Previous groupshave focused on impairing the JAK/STAT3 pathway within tumorcells using in vitro and immunocompromised xenograft modelswith the U87 cell line (37). In contrast, our work utilizes animmunocompetent transgenic murine glioma model that reca-pitulates low- to high-grade glioma transformation. Targetingglioma tumor cells remains the current standard of oncologiccare for patients with malignant glioma; tumor escape mechan-isms harbored within the stromal infrastructure remain an under-targeted aspect of complex malignant glioma biology. Neuro-oncologists lack a wide treatment armamentarium based upontumor-extrinsic therapies beyond early-generation anti-VEGFantibodies. Our in vitro studies with AZD1480 showed no effecton cellular proliferation (Supplementary Fig. S4C and S4D),suggesting a tumor-extrinsic effect of BMDC targeting.

Achieving adequate CNS penetration of agents through theblood brain barrier is also a significant problem for tumor-intrinsic therapies (38, 39). Therefore, new agents that targetspecific BMDCs warrant consideration and discussion. Ultimate-ly, a more comprehensive understanding of themany facets, bothintrinsic and extrinsic, that comprise the molecular complexity oftumor progression may help us to stratify patients into targetedregimens.

Our data demonstrates the peripheral upregulation ofCD11bþ/GR1þ cells within the bone marrow and increased

mobilization into the peripheral blood en route to the tumor site.Similar to peripheral blood data from our murine glioma model,human patients with high-grade gliomas had a higher level ofCD11bþ cells found in the peripheral bloodwhen comparedwiththose with low-grade gliomas (Fig. 1A). We suggest cross-talkoccurs between the tumor andmicroenvironment via tumor cell–secreted factors that lead to tumor progression. More studies arenecessary to define these factors. Understanding the cross-talkbetween the tumor andmicroenvironment (BMDCs), particularlywith respect to how AZD1480 influences the recruitment ofBMDCs during the low- to high-grade transition is paramount.Using our RCAS murine glioma model with GFPþ bone marrowtransplant, we also attempted to sort GFPþ/BMDCsþ to allow usto run a cytokine array comparing cells from AZD1480-treatedand vehicle control animals. However, despite several attempts,the BMDC cellular yield from this brain-derived population wastoo low to permit this type of analysis. It is important to identifythe cytokines, growth factors, and chemokines that may play asignificant role in the recruitment process of BMDCs and subse-quent studies should address this important point. The role ofnon-CD11bþ cells and resident microglia (BMDCþ/GFPnegative)should also be further studied as infiltrating CD11bþ myeloidcells are only 33%of the recruitedGFPþ population in our animalmodel.

To dissect the contribution of BMDCs during low- to high-grade transformation, bone marrow transplant studies were per-formed demonstrating the recruitment of GFPþ BMDCs, whichcorrelated with high tumor burden. We observed a higher quan-tity of CD11bþ cells in both the periphery (blood and bonemarrow) and at the tumor site in high-grade tumor-bearinganimals. We also observed a perivascular distribution of CD11bþ

cells suggesting a role in neovascularization. These results supportprevious extensive reports describing myeloid-associated tumorangiogenesis in cancer (11, 40–43).

After observing BMDC mobilization in our animal model, weattempted to impair recruitment before low-grade glioma trans-formation to mitigate the effects of the angiogenic switch. TheJAK/STAT pathway has been actively studied with respect to itscrucial role regulating myeloid cell development and extravasa-tion. Previous studies in other solid tumors have shown theeffectiveness of AZD1480 in targeting the JAK/STAT3 pathway,including in CD11bþ/GR1þ cells (15, 30). We examined theeffects of AZD1480 on CD11bþ cell recruitment in the low- tohigh-grade murine gliomamodel. We observed reduced CD11bþ

cell levels in the bone marrow, peripheral blood, and at theregional tumor site in AZD1480-treated animals. We also dem-onstrated significant impairments in tumor progression by his-tologic analyses, radiographic findings, and overall survival inanimals treated with AZD1480 compared with vehicle controlmice.While previousfindings in other solid tumormodel systemshavedemonstratedmyeloid cell infiltration, toour knowledge,weare the first to report that this process holds true in early-stagegliomagenesis. Reports have also demonstrated BMDCs (using aCSFR-1 inhibitor) as an important factor that promote angiogen-esis and invasion in high-grade gliomas (44). AZD1480-treatedanimals demonstrated decreased microvascular proliferationwhen compared with vehicle control animals. These data suggestAZD1480mayhave an antiangiogenic effect inourmurine gliomamodel. Limitations of antiangiogenic therapies have beendescribed in the glioma literature including a lack of definitivedata confirming antitumor effect with these agents (45).






Furthermore, inhibiting angiogenesis in high-grade gliomas mayantagonize the efficacy of chemotherapeutic agents by normal-izing the blood brain barrier.

The earliest stages of gliomagenesis represent a unique stageof glioma biology during which the microenvironment may bemore actively evolving than the tumor cells and thus mayrepresent a novel approach to cancer therapy. Currently, gliomaprogression is monitored largely by MRI. More precise tumorsurveillance may both reduce unnecessary treatments forpatients who are not likely to progress and yet simultaneouslyprompt earlier and more appropriate interventions for patientswith low-grade gliomas at the earliest stages of malignanttransformation. Our data demonstrate one important factorin glioma progression: the BMDC recruitment process from thebone marrow. These cells can be easily monitored duringcritical stages of glioma progression and may serve as a bio-marker of low-grade transformation with a simple peripheralblood assay. Utilizing this data along with more traditionalmarkers of progression such as MRI may more accuratelypredict early glioma progression. Studies aimed at collectionof peripheral blood samples of patients with low-grade (gradeII) lesions may help quantify and characterize mobilizedBMDCs. RNA-sequencing assays of this crucial cellular popu-lation may help identify the factors regulating their recruitmentand their interactions within the tumor microenvironment.Appreciating differences in these BMDC populations duringstages of glioma progression may serve as a platform to stratifywhich patients may benefit from early-stage anti-CD11bþ ther-apy and which patients would be better served by observation.Further studies in select glioma patient cohorts utilizing JAK 1/2 inhibitors that are currently used in clinical practice for otherindications warrant further consideration in this unique patientpopulation.

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

Authors' ContributionsConception and design: P. Rajappa, W.S. Cobb, Y. Huang, J.F. Bromberg,J.P. GreenfieldDevelopment of methodology: P. Rajappa, W.S. Cobb, C. Hoffman,J.F. Bromberg, J.P. GreenfieldAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Rajappa, W.S. Cobb, E. Vartanian, J. Zhang,B. Shen, K. Garg, S. Haddock, D.J. Pisapia, T.A. ChanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Rajappa, W.S. Cobb, E. Vartanian, Y. Huang,J. Zhang, K. Garg, B. Cisse, D.J. Pisapia, T.A. Chan, D. Lyden, J.F. Bromberg,J.P. GreenfieldWriting, review, and/or revision of the manuscript: P. Rajappa, W.S. Cobb,E. Vartanian, L. Daly, C. Hoffman, R. Yanowitch, K. Garg, B. Cisse, J.T. Huse,D.J. Pisapia, T.A. Chan, J.F. Bromberg, J.P. GreenfieldAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P. Rajappa, E. Vartanian, L. Daly, K. Garg,J.T. Huse, T.A. ChanStudy supervision: T.A. Chan, D. Lyden, J.F. Bromberg, J.P. Greenfield

AcknowledgmentsWe are grateful to Maria Jiao (MSKCC Center of Comparative Medicine &

Pathology), Kenneth Pitter and the Holland Laboratory, Leila Akkari and theJoyce Laboratory, Dmitri Yarilin (MSKCC Molecular Cytology Core Facility),and the Children's Cancer and Blood Foundation. Our gratitude goes to thefamilies of the Children's Brain Tumor Project for helping us support this work.We would also like to thank Bing He, PhD, from the Translational ResearchProgram at Weill Cornell Medicine.

Grant SupportThis work was funded by the Children's Brain Tumor Project atWeill Cornell

Pediatric Brain and Spine Center (to J.P. Greenfield). This work was alsosupported by grants from the Charles and Marjorie Holloway Foundation(to J. Bromberg), Sussman Family Fund (to J. Bromberg), Lerner Foundation(to J. Bromberg), and the MSK Cancer Center Support Grant/Core Grant (P30CA008748; to J. Bromberg).

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 June 23, 2016; revised December 20, 2016; accepted December 20,2016; published OnlineFirst December 30, 2016.

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2017;23:3109-3119. Published OnlineFirst December 30, 2016.Clin Cancer Res Prajwal Rajappa, William S. Cobb, Emma Vartanian, et al. JAK-mediated Recruitment of Myeloid CellsMalignant Astrocytic Tumor Progression Potentiated by

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