Radiation with STAT3 blockade triggers dendritic cell-T cell … · 2020-06-30 · 1 Radiation with...

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Radiation with STAT3 blockade triggers dendritic cell-T cell interactions in the

glioma microenvironment and therapeutic efficacy

*Martina Ott1, *Cynthia Kassab1, *Anantha Marisetty1, Yuuri Hashimoto1, Jun Wei1, Daniel

Zamler2, Jia-Shiun Leu1, Karl-Heinz Tomaszowski5, Aria Sabbagh1, Dexing Fang1, Pravesh

Gupta8, Waldemar Priebe9, Rafal J. Zielinski9, Jared K. Burks6, James P. Long7, Ling-Yuan

Kong1, Gregory N. Fuller3, John DeGroot4, Erik P. Sulman10, Amy B. Heimberger1

1Departments of Neurosurgery, 2Genomic Medicine, 3Neuropathology, 4Neuro-Oncology,

5Cancer Biology, 6Leukemia, 7Biostatistics, 8Translational Molecular Pathology, 9Experimental

Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030;

10Department of Radiation Oncology, NYU Langone Health Perlmutter Cancer Center, New

York, NY.

*Co-lead authors

Running title: WP1066 + WBRT induce dendritic cell-T cell interaction

Key words: STAT3, dendritic cells, T cells, whole-brain radiation therapy, glioma

Abbreviations: APC, allophycocyanin; BBB, blood-brain barrier; CNS, central nervous system;

EGFR, epidermal growth factor receptor; FITC, fluorescein isothiocyanate; GBM, glioblastoma

multiforme; IFN, interferon; IL, interleukin; LN, lymph nodes; MHC, major histocompatibility

complex; Ova, ovalbumin; PE, phycoerythrin; STAT3, signal transducer and activator of

transcription 3; TNF, tumor necrosis factor; Tregs, regulatory T cells; HBSS, Hanks’ balanced

salt solution; WBRT, whole-brain radiation therapy.

Research. on November 9, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4092

http://clincancerres.aacrjournals.org/

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Funding: This research was supported by the Cancer Prevention and Research Institute of

Texas IIRA-RP160482, the National Institutes of Health CA1208113, P50 CA093459, P50

CA127001 and P30 CA016672, the Ben and Catherine Ivy Foundation, the MD Anderson GBM

Moonshot, and the Brockman Foundation

Requests for reprints: Amy B. Heimberger, MD, Department of Neurosurgery, The University

of Texas MD Anderson Cancer Center, Unit 422, P.O. Box 301402, Houston, TX 77230-1402.

Phone (713) 792-2400, Fax (713) 794-4950, e-mail [email protected].

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed by the authors.

Word count: 5439

Figures: 6

Author Contributions:

Experimental design and/or implementation: MO, YH, AM, JW, DZ, K-H T, CK, JKB, DF, AS,

PG, WP, RJZ, L-Y K, ES, ABH

Analysis and interpretation of the data: MO, YH, JW, DZ, J-SL, CK, JPL, GNF, JD, ES, ABH

Writing of the manuscript: MO, YH, AM, ABH

Have read and approved the final version: MO, YH, AM, JW, DZ, J-SL, K-H T, DF, CK, JKB,

JPL, L-Y K, GNF, JD, ES, ABH



mailto:[email protected]


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Translational Relevance

Given the heterogeneous nature of gliomas, it is unlikely that a monotherapeutic strategy would

induce durable responses across patients. This study combines standard-of-care radiation

therapy and a BBB-penetrant small molecule inhibitor, WP1066, which blocks the transcriptional

activity of the signal transducer and activator of transcription 3 (STAT3), which is currently in

clinical trials (NCT01904123). The combination of radiation and WP1066 demonstrated a

marked therapeutic response in a preclinical glioma mouse model, which was mediated by the

immune system, because since the therapeutic effect is lost in immune-incompetent models. By

using nanostring profiling from both the CNS and the peripheral immune compartments and

immunofluorescence, we found that the combination of WP1066 and radiation induces dendritic

cell-T cell interactions in the glioma microenvironment, which seems to be a requirement for a

fully functional immune response. These data provide a strong rationale for clinical trial

consideration in glioma patients.




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ABSTRACT

BACKGROUND: Patients with central nervous system (CNS) tumors are typically treated with

radiation therapy, but this is not curative and results in the upregulation of phosphorylated signal

transducer and activator of transcription 3 (p-STAT3), which drives invasion, angiogenesis, and

immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3

and whole-brain radiation therapy (WBRT) in a murine model of glioma.

METHODS: C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT,

and treatment with WP1066, a blood-brain barrier (BBB)-penetrant inhibitor of the STAT3

pathway, or the two in combination. The role of the immune system was evaluated using tumor

rechallenge strategies, immune incompetent backgrounds, immunofluorescence, immune

phenotyping of tumor-infiltrating immune cells (via flow cytometry), and nanostring gene

expression analysis of 770 immune-related genes from immune cells, including those directly

isolated from the tumor microenvironment.

RESULTS: The combination of WP1066 and WBRT resulted in long-term survivors and

enhanced median survival time relative to monotherapy in the GL261 glioma model

(combination vs. control p<0.0001). Immunological memory appeared to be induced, because

mice were protected during subsequent tumor rechallenge. The therapeutic effect of the

combination was completely lost in immune incompetent animals. Nanostring analysis and

immunofluorescence revealed immunological reprograming in the CNS tumor microenvironment

specifically affecting dendritic-cell antigen presentation and T cell effector functions.

CONCLUSION: This study indicates that the combination of STAT3 inhibition and WBRT

enhances the therapeutic effect against gliomas in the CNS by inducing dendritic-cell and T cell

interactions in the CNS tumor.




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Introduction

Glioblastoma (GBM) is a rapidly growing and diffusely infiltrating tumor in which patients

typically survive for a median of approximately 15 months (1-3). Despite aggressive treatment

including surgery, chemotherapy, and whole-brain radiation therapy (WBRT) (2,4), the

propensity for tumor recurrence in GBM patients is very high, with little improvement of patient

median survival time (5,6). Hence, it is essential to develop a more applicable adjuvant therapy

combined with radiotherapy to improve the outcomes. The STAT3 pathway is a multipotent

regulator of both gliomagenesis (7) and tumor-mediated immune suppression (8-11). STAT3 is

a transcription factor, activated through phosphorylation induced by a variety of signals in a

variety physiological processes. In the tumor microenvironment, the epidermal growth factor

receptor (EGFR) and the interleukin-6 (IL-6) signaling pathway play important roles in activating

STAT3 (12,13). Aberrant activated STAT3 in cancer cells inhibits the production of

proinflammatory cytokines that support the maturation of dendritic cells and thereby influences

the generation of antigen-dependent T cells (14,15). STAT3 has also been shown to be

abnormally activated in diverse immune cells within the glioma microenvironment such as T

cells, NK- cells, neutrophils, and different myeloid cell populations, resulting in profound immune

suppression. Thus, in the tumor microenvironment, overactive STAT3 creates an immunological

niche supporting tumor cells and preventing immune surveillance.

WBRT can induce a proneural to mesenchymal transition, with associated invasiveness

and resistance to temozolomide. These changes are associated with the activation of the

STAT3 pathway, and this transition could be blocked with upstream STAT3 inhibitors. As such,

STAT3 blockade has been proposed to prevent the emergence of therapy-resistant

mesenchymal GBM tumors at relapse (16). Various studies have shown different effects of

radiation on the STAT3 pathway, depending on the dosing and timing. In a more recent study,

low-dose radiation therapy has been proposed as a way to actually inhibit the STAT3 signaling

pathway (17), whereas others have shown that irradiation with higher doses promotes the




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phosphorylation of STAT3 in a dose- and time-dependent manner (18). Radiation therapy has

also been reported to lead to increased expression of STAT3 in a variety of solid tumors

including melanoma (19,20), lung (21), breast (12), and head and neck tumors (22). However,

radiation can have positive antitumor immune stimulatory effects. For example, damage-

associated molecular patterns such as those of HBMG1 and adenosine triphosphate being

released from dying tumor cells can trigger the maturation of dendritic cells plus antigen uptake,

thereby resulting in T cell action and recruitment to kill specific tumor cells (23,24). Radiation

therapy is also known to induce antigen shedding (25).

WP1066 is blood-brain barrier (BBB)-penetrant caffeic acid analogue that blocks the

nuclear translocation of p-STAT3 (26) and is now being used in a clinical trial (NCT01904123).

WP1066 has demonstrated potent direct cancer cell cytotoxicity against a wide variety cancers

and therapeutic in vivo efficacy against gliomas (7,8,27,28), leukemia (29), melanoma (30-32),

squamous cell cancer (33,34), renal cell cancer (35), non-small cell lung cancer (36), and breast

cancer (37). Notably, many of these malignancies are treated with radiation therapy as a

standard of care. WP1066 also has significant immune-modulatory properties, including on

innate immune cells. Specifically, WP1066 can induce the expression of costimulatory

molecules on peripheral macrophages and tumor-infiltrating microglia ex vivo from glioblastoma

patients, cell types that are typically refractory to modulation with other types of immune

therapeutics. WP1066 treatment of the peripheral blood from glioblastoma patients who are

immunologically anergic triggers marked production of proinflammatory cytokines, induces T cell

proliferation and effector responses, and inhibits regulatory T cells (Tregs) (8). Furthermore, the

immunosuppressive properties of glioblastoma cells are significantly diminished upon treatment

with either siRNA targeting STAT3 or with physiological doses of WP1066 (9,11). Collectively,

these data indicate that WP1066 can reverse both innate and adaptive tumor-mediated immune

suppression and that it has direct antitumor effects. Hence, we hypothesized that abnormal

activation of STAT3 would be a potential therapeutic target in the radiation resistance and that




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STAT3 blockade would be a potent inducer of antitumor immune cytotoxicity. To evaluate

therapeutic synergistic activity and host immune response, an immune-competent glioma model

system was used (38,39). The combined treatment of WP1066 and WBRT demonstrated

increased median survival time, induction of immunological memory, and increased antigen

presentation and T cell activation during the therapeutic window within the CNS glioma

microenvironment.

Methods

In Vivo Murine Tumor Models

All animal experiments were conducted in compliance with the guidelines for animal care and

use established by The University of Texas MD Anderson Cancer Center (MD Anderson) under

the IACUC approved protocol (00001176-RN00). The murine glioma GL261 cell line was

purchased from the NIH. These cells were maintained in Dulbecco’s modified Eagle’s medium

(Life Technologies; Grand Island, NY), supplemented with 10% FBS, 1% penicillin/streptomycin,

and 1% L-glutamine, at 370 C in a humidified atmosphere of 5% CO2 and 95% air. The cells

were cultured and numerically expanded for up to 2 weeks before intracranial implantation and

tested in the week before the injection for Mycoplasma contamination (MycoAlert, Lonza).

To induce intracranial tumors in C57BL/6J or nude mice, GL261 cells were collected in

logarithmic growth phase, loaded into a 25 µL syringe (Hamilton, Reno, NV) and injected 2 mm

to the right of bregma and 4 mm below the surface of the skull at the coronal suture using a

stereotactic frame (Stoelting, Wood Dale, IL). The intracranial tumorigenic dose for GL261 cells

was 5 x 104 in a total volume of 2 μl. Mice were randomly assigned to control or treatment

groups (n=6-10/group) after tumor implantation for the intracranial model systems. The animals

were observed daily, and when they showed signs of neurological deficit (lethargy, failure to

ambulate, lack of feeding, or loss of >20% body weight), they were compassionately killed.




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These symptoms typically occurred within 48 hours before death. Their brains were removed

and placed in 4% paraformaldehyde and embedded in paraffin.

Treatments

WP1066 (26), which blocks p-STAT3 (8), was synthesized and supplied by Dr. Waldemar

Priebe (of MD Anderson). WP1066 does not influence JAK2 kinase activity at concentrations up

to 10 µM based on KINOME scan profiling (Supplementary Table 1), and this compound has a

selectivity score S (35) of 0.037. The IC50 of WP1066 for GL261 is 4.91 µM (Supplementary

Fig.1A), whereas immune modulation occurs at 1 µM (8,31). For in vivo treatment, the mice

were treated via oral gavage with WP1066 (60mg/kg) in a vehicle of DMSO/PEG300 (20

parts/80 parts) or vehicle control on a Monday/Wednesday/Friday schedule for 3 weeks, starting

on day 7 after glioma implantation when gliomas have been shown to be established in the

brain (40) and consistent with the treatment window used for evaluating other

immunotherapeutics in this model (41,42), which results in a serum circulating level in the range

of approximately 1-3 µM (Supplementary Fig. 1B). For WBRT, mice were anesthetized using

isofluorane, and the whole brain was irradiated at a 2 Gy dose with an opposing lateral plan

using a 15 mm collimator. The dosing and schedule of the radiation were optimized so as to not

be curative, thus enabling an assessment of whether an additive or synergistic effect could be

detected (Supplementary Fig. 2).

IC50 Cell Proliferation/Survival Assay

GL261 cells were seeded in triplicate at a density of 2,000 cells per well in 96-well culture plates

and were treated with WP1066 at increasing concentrations of 0, 1.56, 3.13, 6.25, 12.5, and 25

μM. After 72 h of treatment, 25 ml of 5 mg/ml dimethyl thiazolyl diphenyl tetrazolium salt (MTT,

Sigma-Aldrich, St. Louis, MO) solution was added to each well, and the cells were cultured for 3

h at 37° C in a humidified atmosphere of 5% CO2 and 95% air. The cells were lysed with 100




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μl/well of lysing buffer (50% dimethylformamide, 20% SDS, pH 5.6) and incubated at room

temperature overnight. Cell proliferation and viability were evaluated by reading the O.D. at 570

nm, and the IC50 was calculated.

Magnetic Resonance Imaging (MRI)

Mice were imaged at the MD Anderson Small Animal Imaging Facility using a 7 Tesla (T) 30-cm

horizontal bore magnet (Bruker Biospin MRI, Billerica MA). Each mouse was anesthetized with

2% isoflurane during imaging. For tumor detection, T2-weighted images (21 transverse slices

with a thickness of 0.75 mm and taken in a field of view [FOV] of 30 x 22.5, with a matrix size of

256 x 192 pixels, for a resulting in-plane resolution of 0.117 µm) were acquired using a RARE

(rapid acquisition with relaxation enhancement) sequence, with a repetition time (TR) of 3000

ms and an echo time (TE) of 57 ms. The tumor volume was determined by using the software

ImageJ.

HPLC Detection and Quantification of Serum Concentration of WP1066

Mice were treated via oral gavage with WP1066 (60mg/kg) on Monday, Wednesday, and

Friday, and blood was collected via terminal cardiac puncture at various time points into

K2EDTA blood vacutainers (BD Bioscience): Day 1: 0h (pretreatment), 0.5h, 1h, 2h, 4h, 8h,

12h; Day 2: 0h, 0.5h, 1h, 2h, 12h; Day 8: 0h (pretreatment), 0.5h, 1h, 2h, 4h, 8h, 12h; and Day

9: 0h, 0.5h, 1h, 2h, 12h. The blood was centrifuged (1000 g, 15 min, 4°C) and the plasma

immediately transferred and frozen (-70°C or below). A validated method for detecting the

concentration of WP1066 was conducted by IIT Research Institute (Chicago, IL).

KINOMEscan TM Profiling of WP1066 – scanMAX




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KINOMEscan profiling of WP1066 at concentrations of 1 µM and 10 µM were assessed using

Assay scanMAX performed by DiscoverX (https://www.discoverx.com/services/drug-discovery-

development-services/kinase-profiling/kinomescan).

In Vivo Experiment for NanoString Gene Expression Analysis

GL261 cells were injected intracranially into C57BL/6 mice and treated as described above.

MRIs were taken at various time points to verify the presence of tumor. On day 15, the mice

were euthanized, and their spleens were removed and their brains collected after cardiac

perfusion with PBS. Immune cells were isolated from the brains by Percoll gradient density

centrifugation, followed by an “untouched” T cell selection (Mouse Pan T Cell Isolation Kit II,

MACS Miltenyi Biotec). Afterwards, RNA was isolated from both the T cell fraction and the flow-

through non-T cell fraction (CD11b+, CD11c+, CD19+, CD45R+, CD49b+, CD105+, MHC class

II+, Ter-119+), which would include the antigen-presenting cells (RNeasy Plus Mini Kit, Qiagen)

for NanoString gene expression analysis. For the characterization of the T cell fraction and the

flow through, the non-T cell fraction (other immune cells) was first stained with fixable viability

dye efluor 780 to exclude dead cells (Thermo Fisher Scientific), followed by staining with anti-

mouse CD45 BV510 (clone 30-F11), anti-mouse CD3 PerCP.Cy5.5 (clone 17A2), anti-mouse

CD11b PE (clone M1/70), anti-mouse CD11c APC (clone N418), anti-mouse CD19 BV421

(clone 6D5), anti-mouse CD49b PE/Cy7 (clone DX5) (all Biolegend). Afterwards, cells were

fixed with fixation buffer (BD Bioscience) and measured using FACS Celesta (BD Bioscience).

The data analysis was done with FlowJo software.

Ex Vivo Flow Analysis of Tumor-Infiltrating Immune Cells

GL261 cells were injected intracranially into C57BL/6 mice and treated as described above. On

day 17, the mice were euthanized, and their brains were collected after cardiac perfusion with

PBS. Immune cells were isolated from the brains by Percoll gradient density centrifugation.



https://www.discoverx.com/services/drug-discovery-development-services/kinase-profiling/kinomescan

https://www.discoverx.com/services/drug-discovery-development-services/kinase-profiling/kinomescan


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Cells were then first stained with fixable viability dye efluor 780 to exclude dead cells (Thermo

Fisher Scientific). For the distinction of dendritic cells and microglia, cells were stained with anti-

mouse CD45 BV421 (clone 30-F11), anti-mouse CD11c APC (clone N418), anti-mouse CD103

BV605 (clone 2E7), anti-mouse CD11b PE (clone M1/70), anti-mouse MHC-II BV785 (clone

M5/114.15.2) (all Biolegend), and anti-mouse Tmem119 (28-3; abcam), followed by staining

with the secondary goat anti-rabbit AlexFluor488 antibody. Afterwards, cells were fixed with

fixation buffer (BD Bioscience) and measured using FACS Fortessa (BD Bioscience). The data

analysis was done with FlowJo software.

NanoString

RNA (200 ng) at a concentration of 40 ng/μl in a total volume of 5 μl was prepared for

NanoString assay analysis with the immune-specific gene array kit (NanoString Technologies,

Inc.). Sample preparation and hybridization were performed for the assay according to the

manufacturer’s instructions. Briefly, RNA samples were prepared by ligating a specific DNA tag

(mRNA-tag) onto the 3' end of each mature mRNA, and excess tags were removed via

restriction enzyme digestion at 37°C. After processing using the mRNA sample preparation kit,

the entire 10-μl reaction volume containing mRNA and tagged mRNAs was hybridized with a

10-μl reporter CodeSet, 10 μl of hybridization buffer, and a 5-μl capture ProbeSet (for a total

reaction volume of 35 μl) at 65°C for 16-20 hours. Excess probes were removed using two-step

magnetic bead-based purification with an nCounter Prep Station. The specific target molecules

were quantified using an nCounter Digital Analyzer by counting the individual fluorescent bar

codes and assessing target molecules. The data were collected using the nCounter Digital

Analyzer after obtaining images of the immobilized fluorescent reporters in the sample cartridge

using a charge-coupled device camera. These data were then normalized to mRNA gene

expression data for all 770 immune-related genes using the NanoStringNorm R package

(version 1.1.17). The cluster analyses were used to determine deregulated genes between




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different treatment and control groups by multigroup comparison using Qlucore software (Lund,

Sweden). The genes specific to certain immune cell types and functional signaling pathways

were categorized based on the attached kit manual.

Gene Set Enrichment Analysis (GSEA)

Data from the NanoString experiment were loaded into an nCounter (Nanostring Technologies)

to generate corrected counts using internal standards. For each of the categories provided from

the Nanostring manual, a gene set was generated. Corrected counts were then loaded using

software GSEA 4.0.3 for all four samples, and the GSEA analyses were run on all gene sets.

These were used as our gene set database with 1000 permutations and with no gene collapsing

or remapping. Heatmaps, enrichment scores, and correlations were all produced in GSEA (43).

The analysis failed for some of the gene sets when the gene set was either too small or did not

have enough genes expressed in the dataset.

Immunofluorescence

A tyramide signal amplification protocol was used to show the CD11c expression in the brain

tumors. The CD11c antibody (abcam ab219799) was validated using immunohistochemistry.

Perkin Elmer DAPI diluted to 1:75 was used as a nuclear counterstain. Slides were incubated

for 2 h at 60°C, dewaxed in xylene (3 times x 10 min), and rehydrated through a graded series

of ethanol solutions 100% (2 times x 5 min), 90% (2 times x 5 min), 80% (1 time x 5 min), 70%

(1 time x 5 min) followed by a PBS rinse (2 times x 5 min). The slides were fixed with hydrogen

peroxide and methanol (3% w/v) for 20 min at room temperature then rinsed again with PBS (2

times x 5 min). Antigen retrieval was done using the BioGenex EZ-Retriever System V.3

microwave for 1 cycle of 15 min at 95°C with a pH 9 buffer. Slides were left to cool for 30

minutes and then rinsed again in PBS (2 x 5 min). Perkin Elmer ready to use blocking solution

was applied for 35 minutes, and then the slides were incubated with the primary antibody diluted




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in the same blocking solution (1:100) overnight. The second day, the slides were washed with

PBS mixed with 0.1% Tween 20 (TPBS) (3 times x 5 min). Then slides were incubated in the

Perkin Elmer HRP polymer solution for 30 minutes at room temperature (approx. 2-3 drops per

slide), then washed again with TPBS (3 x 5 min). Slides were incubated in the fluorochrome

solution (Opal reagent 570 diluted with the amplification solution to 1:100) for 5 minutes at room

temperature, and then washed with TPBS (3 x 5 min). Perkin Elmer DAPI diluted in PBS to 1:75

was used as a nuclear counterstain for 15 minutes at room temperature, and then slides were

washed with PBS (1 x 5 min) and mounted with Dako fluorescence mounting medium and 22 x

50 No 1.5 thickness coverslip glass. Slides were air-dried, labeled, and stored at 4°C.

For dual CD3 and CD11c immunofluorescence analysis, slides were baked in the oven

at 65⁰C for 1 hour, dewaxed with xylene (3 times x 10 min) and rehydrated through a graded

series of ethanol solutions (100% 1 x 10 min; 95% 1 x 10 min; and rinse in 70%). After

rehydration, slides were rinsed in distilled water and were fixed with 1% H2O2 in 10% methanol

for 15 minutes. Slides were then rinsed in distilled water and then in the appropriate pH 9

antigen retrieval buffer for the CD11c marker. Slides were treated in the EZ Retriever

microwave for 15 minutes at 95°C, and then were left to cool down at room temperature (15 –

30 min). Slides were rinsed in distilled water followed by TPBS (1%). Blocking was done using

Dako ready-to-use reagent. The primary antibody CD11c (abcam, ab219799, 1:75) was added

on to the slides and incubated overnight at 4°C. Slides were rinsed in TPBS 3 times x 2 min at

room temperature. Slides were incubated in Perkin Elmer Polymer horse radish peroxidase for

mouse and rabbit (HRP Ms + Rb) for 20 min at room temperature then rinsed again with TPBS

3 times x 2 min followed by incubation for 6 minutes with Opal Fluorophore Working Solution on

each slide (fluorophore 570, 1:100), and then rinsed with TPBS 3 times x 2 min. The process

was repeated again for the addition of the second antibody (CD3; abcam, ab16669, 1:600).

Slides were treated again with the EZ retriever microwave for 1 cycle of 15 minutes at 95°C with

Agilent pH 6 Ag retriever buffer. Microwave stripping was followed by the same steps as above,




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with the washing in between: blocking, addition of the CD3 antibody (abcam, ab16669, 1:600,

overnight at 4°C), HRP treatment, and Opal 690 fluorophore diluted 1:100. Finally, slides were

counterstained with DAPI (10 µl in 5ml PBS) for 15min, and then they were mounted with DAKO

fluorescence mounting medium.

The dual staining was quantified manually. Slides were scanned with the Vectra Polaris,

and fields of interest containing the tumor were stamped in Phenochart. The positives were

sorted into 2 categories: dyads of one CD3+ cell interacting with one CD11c+ cell within a

distance of 15 nm and clusters defined by two or more CD11c+ cells and two or more CD3+

cells. Data were merged in ExCel, analyzed for significance level, and plotted.

Immunohistochemistry for p-STAT3

Formalin-fixed, paraffin-embedded brain tumor slides were incubated for 1 hour at 60°C,

dewaxed in xylene, and rehydrated in a graded ethanol series (100%, 95%, 70%) followed by

water. Antigen retrieval was performed using a citrate buffer at pH9 in a pressure cooker at

120°C for 12 minutes, followed by two washes in 1 x TPBS buffer mixed with 0.1% Tween 20 for

5 minutes. Peroxidase activity was blocked with 10% methanol and 1% hydrogen peroxide for

30 minutes followed by a wash. The slides were then blocked with protein blocker (Dako) for 15

minutes. The primary antibody was added to the slides and incubated overnight at 4°C (pStat3,

abcam: ab76315). Three washes were done followed by incubation with the secondary antibody

for 30 minutes at room temperature. The DAKO DAB kit was used for color development (10-60

sec depending on the antibody), then counterstained with hematoxylin (25 seconds) and bluing

buffer, rehydrated, and cover-slipped.

Statistical Analysis

Kaplan-Meier product-limit survival probability estimates of overall survival were calculated (44),

and log-rank tests (45) were performed to compare overall survival between treatment groups




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and the control arm. To compare the amount of dyads and clusters between the different

treatment groups, the mean number of dyads and cluster per field were computed. Two-sample

t tests were performed on the mean number of dyads and mean number of clusters among

treatment groups.

Results

STAT3 Pathway Blockade in Combination with WBRT Increases the Incidence of Long-Term

Survivors in Mice with Intracerebral Gliomas

C57BL/6J mice bearing intracranial GL261 tumors were treated with WBRT and/or WP1066.

The mice were randomized to receive: 1) WP1066 (60 mg/kg for 2 weeks on M, W, F); 2)

radiation at 2 Gy; 3) radiation + WP1066; and 4) no treatment/control (Fig. 1A). Subtherapeutic

doses of WP1066 were used to look for synergy with irradiation. WP1066 treatment was started

on day 7 after tumor implantation, and WBRT was administered on day 10 (Fig. 1A). In two

different experiments (Supplementary Fig. 3), long-term durable survival was observed only

with the combination of radiation and WP1066 (Fig. 1B). More specifically, the control mice had

a median survival time of 23 days, WP1066-treated mice had a median survival time of 27 days

(p= 0.1178 versus control), WBRT-treated mice had a median survival time of 28 days (p =

0.0320 versus control) and WBRT + WP1066-treated mice had a median survival time of 32

days. And notably, 40% were long-term survivors (>60 days after tumor implantation), which

was statistically significant relative to controls (p <0.0001), WP1066 monotherapy (p = 0.0004),

and WBRT alone (p = 0.0035). Magnetic resonance imaging (MRI) on day 17 after tumor

implantation confirmed that tumors were present in all groups. Four of 7 imaged mice from the

combined treatment group showed very low tumor burden compared with the other groups,

indicating that the combination therapy was suppressing tumor growth. (Fig. 1C). In the

combined treatment group, the long-term survivors did not show evidence of persistent tumor on

MRI (Fig. 1D). Rechallenge of the tumors in the contralateral hemisphere demonstrated




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protective immunity (Fig. 1E). Immunohistochemical analysis of p-STAT3 during the therapeutic

window (Supplementary Fig. 4) showed reduced staining intensity for p-STAT3, especially in

the WBRT + WP1066-treated mice.

Therapeutic Activity of STAT3 Pathway Blockade in Combination with WBRT Requires the

Immune System

To ascertain whether the therapeutic effect of the combination required an intact immune

system, the prior therapeutic experiment was repeated using nude (athymic) mice (Fig. 1F). An

equivalent number of treatment/cycles were administered to the nude mice relative to the

immune-competent model, and treatment failed to demonstrate a therapeutic response (Fig.

1G), suggesting that the immune system has a mechanistic role in the group receiving the

combined therapy. As such, additional analysis was conducted to verify the role of the immune

system.

WP1066 Combined with WBRT Modulates Immune Responses Directly in the CNS Glioma

Microenvironment

Because we had evidence that the immune system was required for response to the

combination therapy, we next evaluated alterations in immune responses in both the peripheral

and glioma microenvironment. Day 15 was selected for the therapeutic window analysis (Fig.

2A) after we documented that the tumors were large enough to analyze (Fig. 1C) but before the

animals were moribund and treatment was failing (Fig. 1B). The T cells (CD3+) and other

immune cells (CD11b+, CD11c+, CD19+, CD45R+, CD49b, CD105+, MHCII+, Ter-119) were

isolated from the brains and spleens, respectively, that were pooled from 3-4 mice from groups

that were either untreated, treated with WP1066, WBRT, or WP1066 + WBRT. T cell purity was

approximately 73%, and the other immune cells consisted mostly of CD11b+




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monocyte/macrophages and CD11c+ dendritic cells (Fig. 2B). To screen for mechanisms that

could contribute to the observed survival benefit mediated by the combinatorial treatment, gene

expression analysis was performed using NanoString, which profiled a total of 770 genes

(Supplementary Fig. 5). When we compared the immunological responses of the various

treatment groups, we were surprised to find that the most robust immunological responses,

such as interferon-induced-responses, IL-1-associated genes, and the toll-like receptor pathway

were most robustly upregulated in the brain’s immune cells rather than in the peripheral spleen

cells (Fig. 2C). Peripheral immune monitoring has not been correlated with therapeutic

responses before, and these data suggest that the brain compartment may be a more

appropriate location for monitoring antitumor immune responses. As such, we focused our

analysis on the immune cell responses and interactions within the brain.

WP1066 Combined with WBRT Globally Reprograms the Immune Responses in the Tumor

Microenvironment

Immune gene sets were annotated and categorized based on their known functions. For each of

these categories, we performed Gene Set Enrichment Analysis (GSEA) to compare the

combinatorial treatment group with the control and monotherapy groups. For both data sets, (T

cells [Fig. 3A] and other immune cells [Fig. 3B]), the normalized enrichment score (NES) for

each gene set was ranked from high to low in order to clarify the mechanisms that could have a

significant influence in the response to the treatment combination. For example, genes

associated with the generalized functions of immune cells (such as autophagy, cancer

progression, the cell cycle, senescence) and genes associated with specific immune cell

populations such as Tregs, B cells, Th2 cells, and NK cells, were not enriched in the

combination cohort, which indicated that there is specificity in the immune functions that are

upregulated in the glioma microenvironment (Fig. 3, Supplementary Fig. 6). Whereas within

the significantly enriched gene set, many were associated with T cell-dendritic cell interactions




18

such as antigen processing, MHC-I/-II, dendritic cell function, phagocytosis, and T cell

proliferation. Indeed, the only gene set significantly enriched in the other immune cell population

was dendritic cell function (Fig. 3B, C). These data suggest that antigen presentation is a

requirement within the tumor microenvironment of the CNS for full antitumor immune-mediated

activities mediated by the combination of WP1066 and WBRT.

WP1066 Combined with WBRT Triggers Dendritic Cell-T cell Immunological Interactions in the

Glioma Microenvironment

Dendritic cells usually reside in lymphoid organs, and their presence in gliomas has only

recently been noted (46). The NanoString analysis strongly implicated this immune cell

population in the therapeutic activity of the combination in the glioma microenvironment. To

validate this key NanoString finding, we performed immunohistochemical analysis of the glioma

during the therapeutic window. Glioma-bearing mice (n=16) were either untreated (n=4) or

treated with WP1066 (n=4), WBRT (n=4), or the two in combination (n=4). Immunofluorescence

detection for CD11c+ dendritic cells demonstrated that these cells are abundant at the invasive

edge (Fig. 4A) and are diffusely present throughout the glioma (Fig. 4B). Two of 4 mice in the

WP1066 plus WBRT combination group achieved high CD11c positivity (defined as greater than

20% mean positivity), whereas the rates for the other groups were: control group, 0/4; WBRT,

1/4; and WP1066, 0/4 (Fig. 4B, C), further validating the results in the NanoString dataset

indicating that the dendritic cells were markedly enriched in the glioma microenvironment in

mice treated with WP1066 + WBRT.

To ascertain whether the T cells and dendritic cells are directly interacting in the glioma

microenvironment, as would be implied by the NanoString analysis, we performed dual

fluorescence immunohistochemical analysis for both immune populations (Supplementary Fig.

7). A positive interaction was scored when these immune cells were within a 15-nM distance of

each, indicating that the antigen-presenting dendritic cell is triggering T cell activation (47,48).




19

Antigen-specific immune responses are also known to occur in clusters of dendritic cells and T

cells, which are required for T cell proliferation and effector responses (49). Tight dendritic-T cell

intercellular interactions between individual cells (dyads) (Video 1; Fig. 5A, B, C) were much

more frequently observed in all three treatment groups than in the control (Fig. 5E, F), indicating

immunologically synapses were occurring. In addition to dyad formation, clustering interactions

(Video 2; Fig. 5D) were observed almost exclusively in the combinatorial treatment group (Fig.

5E, G) (Control vs. WP1066 p= 0.5696; Control vs. WBRT p= 0.6951; Control vs. WBRT +

WP1066 p= 0.0188; WP1066 vs. WBRT p= 0.3256; WP1066 vs. WBRT + WP1066 p= 0.0140;

WBRT vs. WBRT + WP1066 p= 0.0261).

To further show that the CD11c cells were dendritic cells and not microglia, immune cells

were isolated from all treatment groups during the therapeutic window and analyzed via

multicolor flow cytometry. Dendritic cells (defined as CD45+, CD11c+, MHC-II+, and CD103+ or

CD11b+, Tmem119 negative) were almost exclusively in the CD45 high positive population

(Fig. 6A); whereas microglia (defined as CD45+ CD11b+ Tmem119+) were only detected in the

CD45 intermediate population (Fig. 6B). Direct comparison between immune cells isolated from

naïve brains (Fig. 6B) and the WP1066 + WBRT treated GL261 tumor-bearing brains (Fig. 6A)

showed a marked increase of dendritic cells. Back gating of CD11c+ cells further confirmed that

CD11c+ cells are not microglia cells (Fig. 6C). Quantification of CD103+ CD11c+ CD45 high

dendritic cells, which have been described as a rare population but one remarkably capable of

stimulating cytotoxic T cell responses (50) in the tumor microenvironment, demonstrated a

significant increase in the different treatment groups compared with the control, with the

strongest increase being in the combination group (Fig. 6D). Cumulatively, these data indicate

that dendritic cell-T cell interactions in the glioma microenvironment are contributing to the

therapeutic effect of this particular combination.

DISCUSSION




20

Our NanoString and immunofluorescence in vivo data indicate that the combination of

WBRT and STAT3 inhibition triggers dendritic cell and T cell interactions in the glioma

microenvironment, including antigen presentation and T cell activation—a first report to the best

of our knowledge. Whereas both monotherapies increased singular dendritic cell-T cell

interactions in the tumor microenvironment by an amount similar to what was seen with the

combinatorial treatment, extensive clustering of dendritic cells and T cells (which is an early

event during immune activation) covering large parts of the whole tumor area, was uniquely

observed only in the tumor microenvironment of the combinatorial treatment group, which is

probably the key factor in the therapeutic effect. During the initial antigen presentation events,

immune checkpoints are not yet significantly upregulated (51), and thus T cells are free to exert

their effector responses, including eradication of the tumor. We postulate that if the antitumor

immune activation events only occur in the peripheral lymph node, especially repeatedly and

chronically, with subsequent trafficking of the effector cell to a profoundly immune--suppressive

CNS glioma microenvironment, that the T cell is rendered exhausted and unable to recover

effector functions (52). This immune suppression is especially problematic when STAT3 is

activated, because it is the key transcriptional factor that renders tumor-infiltrating dendritic cells

dysfunctional (53). Because WBRT triggers the activation and influx of the dendritic cells

(54),(55,56), and because STAT3 blockade reverses the immune-suppressive glioma

microenvironment (8,11,28), de novo antigen presentation and T cell activation can occur

unencumbered in this scenario. Alternatively, the observed dendritic cell and T cell interactions

may represent and support the notion of the second-touch hypothesis. In 2014, Klaus Ley put

forth the second-touch hypothesis stating that full T cell activation requires a second interaction

with an antigen-presenting cell in the non-lymphoid, antigen-expressing target tissue (57). This

initially marginalized concept seems to be supported by the findings in this manuscript.

It is unlikely, given the marked heterogeneity of tumors, that single monotherapeutic

strategies will provide uniform, consistent therapeutic responses among all patients. A rational




21

strategy is to build upon standard-of-care treatment strategies such as radiation therapy. Our

preclinical data support clinical trial consideration of the combination of radiation with a STAT3

inhibitor in glioma patients. But even more importantly, our study demonstrated a unique

mechanism of activity—global reprogramming of the immune system within the tumor

microenvironment. Immune clearance of a tumor is not mediated by a single immune cell

population, chemokine, cytokine, or pathway. Rather the combinatorial treatment impacts

different immune cell populations including the CD11c+ dendritic cell, which has not been fully

appreciated previously in the glioma microenvironment. It is through global immunological

reprogramming, that we are able to observe the final end result of increased dendritic cell

infiltration, maturation, and T cell interaction. Future studies will be directed toward further

analysis of the immunological synapse, including further defining of the participating T cell and

dendritic cell subsets. Although we observed significant dendritic cell-T cell interactions in the

combinatorial treatment group, we did not resolve whether these interactions are tumor-antigen

specific. The lack of known tumor antigens in mouse gliomas and the availability of specific

tetramers makes this a challenge for the entire field. Although there are several antigen models

available, they are highly immunogenic, which is not reflective of the immunobiology of gliomas.

Our immune-monitoring data in the preclinical models strongly suggest not only that

peripheral immune monitoring would be of limited utility in the context of human clinical trials but

also that this needs to be done with consideration of the actual tumor microenvironment. An

expansion phase 0/window-of-opportunity cohort should be considered to verify that radiation

therapy plus WP1066 also induces dendritic cell trafficking, maturation, antigen presentation,

and T cell activation/cytotoxicity during the therapeutic window in the context of treated human

subjects. This is a plausible strategy in that we have performed similar studies in glioblastoma

patients receiving immune checkpoint inhibitors prior to surgical resection. A number of

interesting therapeutic targets were also found to be upregulated in this study after




22

administration of the combination of WP1066 and WBRT, including CD274 (PD-L1), which could

also be considered in the future for clinical trial implementation.

Acknowledgments

The authors acknowledge the Flow Cytometry and Cellular Imaging Core Facility at MD

Anderson funded by the National Cancer Institute # CA16672 for their assistance with flow

cytometry data acquisition, the Small Animal Imaging Facility at MD Anderson supported by the

NIH/NCI under award number P30CA016672, and David M. Wildrick, Ph.D., Jennifer Everts,

and Audria Patrick for their editorial and administrative support.

.




23

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30

FIGURE LEGENDS:

Fig. 1: Whole-brain radiation therapy (WBRT) combined with STAT3 inhibitor, WP1066, in the

murine glioma model. (A) Schema of the treatment of immune competent mice that underwent

intracerebral (i.c.) implantation of GL261 glioma cells. Seven days after GL261 implantation,

mice were treated with WP1066 (60 mg/kg) by oral gavage (o.g.) 3 times per week (Monday/

Wednesday/ Friday) for 3 weeks. On day 10, mice received WBRT (2 Gy). Long-term survivors

(>60 days) were rechallenged with GL261 cells in the contralateral hemisphere. (B) Combined

survival curves from two independent experiments. The survival rate of C57BL/6 mice was

estimated by the Kaplan-Meier method. Control: 19 mice (MS:23d), WP1066: 17 mice (MS:27d),

WBRT: 19 mice (MS:28; 1 long-term survivor), WP1066 + WBRT: 20 mice (MS:32.5d; 7 long-

term survivors). Statistics: Control vs. WP1066 p=0.1175; Control vs. WBRT p=0.032; Control

vs. WP1066 + WBRT p<0.0001; WP1066 vs. WP1066 + WBRT p= 0.0004; WBRT vs. WP1066

+ WBRT p=0.0035; WBRT vs. WP1066 p=0.5078. (C) MRI volumetric analysis of 5-7 mice per

treatment group (left), representative magnetic resonance images (MRIs) of the brains of mice

harboring GL261 in each experimental group (right). (D) Representative MRI of a long-term

survivor mouse with GL261 implanted and treated with the combination of WP1066 and WBRT.

(E) Kaplan-Meier survival curves of the rechallenged long-term survivor and naïve control mice.

(F) Schema of the treatment of immune incompetent mice that were intracerebrally implanted

with GL261 glioma cells. Seven days after GL261 implantation, mice were treated with WP1066

(60 mg/kg) 3 times per week (Monday/ Wednesday/ Friday) for 3 weeks and received WBRT (2

Gy) on day 10. (G) The survival rate of nude mice estimated by the Kaplan-Meier method (n=

10/group).

Fig. 2: Heatmaps of NanoString profiling of immune populations from the brains and spleens of

glioma-bearing mice treated with WBRT, WP1066, or the combination. (A) During the

therapeutic window, mice were terminated on day 15, their immune cell populations were




31

purified, the RNA was isolated from them, and then NanoString profiling of 770 genes was

performed. (B) Immune cell purity based on flow cytometry after T cell enrichment. Other

immune cells consist mostly of CD11b+ monocyte/macrophages and CD11c+ dendritic cells.

Some immune cells are positive for several markers, e.g., some of the CD11c+ cells are also

CD11b+. A small percentage of cells were not positive for any of the analyzed markers. (C)

Heat maps demonstrating that for many key immunological effector functions, the brain immune

cells had a higher expression of genes associated with IFN, IL-1, and the toll-like receptor

pathway relative to the peripheral immune cells. o.g., oral gavage.

Fig. 3: Normalized Enrichment Scores (NES) of NanoString profiling of T cells (A) and other

immune cells (B) of immune gene sets in the combinatorial group compared with the control and

monotherapies. Gene sets that are significantly enriched in the combinatorial treatment group

(nominal p value ≤ 0.05) are shown in red. T cells: phagocytosis p ≤ 0.001; MHC-I-II p=0.002;

antigen processing p=0.008; dendritic cell function p=0.023, bacterial response p=0.019;

transporter function p=0.001; T cell proliferation p= 0.026; regulation of inflammatory response

p=0.041; CD molecules p=0.006. Other immune cells: dendritic cell function p=0.049. (C) GSEA

blot and heatmap for the dendritic cell function gene set from the other immune cell populations.

DC, dendritic cells.

Fig. 4: (A) Whole-mount coronal section of GL261 bearing brain immunofluorescently stained

for CD11c+ dendritic cells (green) at 1.5x magnification. DAPI (blue) is used to stain nuclei.

Coronal section is outlined by dashed line. Arrows denote tumor margins. Gliomas were

analyzed 17 days after implantation. (B) Representative GL261 staining for CD11c+ dendritic

cells across treatment cohorts at 40x. (C) Violin plot summarizing the data quantifying CD11c+

expression within GL261 gliomas that were either untreated or treated with WBRT, WP1066, or




32

the combination. Average percentage of CD11c+ positive cells per field: control = 5.53%,

WP1066 = 5.6%, WBRT = 10.6%, WBRT + WP1066 = 16.94%.

Fig. 5: (A) Coronal section of GL261 tumor-bearing brain treated with the combination of

WP1066 and WBRT. The brain was harvested 17 days post implantation and stained for

CD11c+ dendritic cells (green) and CD3+ T cells (red). DAPI (blue) is used to stain the nuclei.

(A, B, C) Immunological synapses between CD11c+ dendritic cells and CD3+ T cells (dyad). (D)

Cluster of CD11c+ and CD3+ T cell interactions (cluster). (E) Representative GL261 staining for

CD11c+ dendritic cells and CD3+ T cells across treatment cohorts at 40x. (F) Box plots

summarizing the data quantifying the number of CD11c+ and CD3+ T cell interactions occurring

in GL261 glioma controls or treated with WBRT, WP1066, or the combination. Interactions were

classified into dyad (one CD11c+ dendritic cell interacting with one CD3+ T cell): Control vs.

WP1066 p= 0.0322; Control vs. WBRT p= 0.1928; Control vs. WBRT + WP1066 p= 0.1124;

WP1066 vs. WBRT p= 0.5887; WP1066 vs. WBRT + WP1066 p= 0.4793; WBRT vs. WBRT +

WP1066 p= 0.9392, and (G) cluster (interaction of at least two dendritic cells with two CD3+ T

cells): Control vs. WP1066 p= 0.5696; Control vs. WBRT p= 0.6951; Control vs. WBRT +

WP1066 p= 0.0188; WP1066 vs. WBRT p= 0.3256; WP1066 vs. WBRT + WP1066 p= 0.0140;

WBRT vs. WBRT + WP1066 p= 0.0261. To compare the amount of dyads and clusters between

the different treatment groups, the mean number of dyads and clusters per field were computed,

and a two-sample t tests were performed across treatment groups.

Fig. 6: Flow cytometry analysis of immune cells isolated from the brain of a mouse treated with

WBRT + WP1066 during the therapeutic window (day 17) (A) and from a naïve non-tumor-

bearing brain (B) to distinguish between dendritic cells (CD45high, CD11c, MHC-II+, and

CD103+ or CD11b+) and microglia (CD45intermediate, CD11b+, Tmem119+). (C) Back gating

of CD11c+ cells (shown in red) isolated from the brain of a mouse treated with WBRT +




33

WP1066. (D) Quantification of CD103+ dendritic cells in the different treatment groups (n=4-

5/group). Control vs WP1066: p=0.0037; control vs WBRT p=0.0096; control vs WP1066 +

WBRT p= 0.0018, as assessed by two-sided unpaired t test.




A B

C

WP1066+WBRT

D

F G

E

0 1 5 3 0 4 5 6 0 7 5

0

5 0

1 0 0

D a y s p o s t G L 2 6 1 c e ll in je c t io n

Pe

rc

en

t s

urv

iva

l

n a iv e m ic e

re c h a lle n g e d m ic e

(W P 1 0 6 6 + X R T )

0 1 5 3 0 4 5 6 0

0

5 0

1 0 0

C o p y o f A ll


Pe

rc

en

t s

urv

iva

l

C o n tro l

W P 1 0 6 6 (6 0 m g /k g )

W B R T (2 G y x 1 )

W P 1 0 6 6 + W B R T

0 1 5 3 0 4 5 6 0

0

5 0

1 0 0

C o p y o f A ll


Pe

rc

en

t s

urv

iva

l

C o n tro l

W P 1 0 6 6 (6 0 m g /k g )

W B R T (2 G y x 1 )

W P 1 0 6 6 + W B R T

0 1 5 3 0 4 5 6 0 7 5

0

5 0

1 0 0

R e c h a lle n g e c o m b in e d


Pe

rc

en

t s

urv

iva

l

n a iv e m ic e

re c h a lle n g e d m ic e

(W P 1 0 6 6 + W B R T )

Figure 1

Control WP1066

WBRT WP1066+WBRT




Isolated immune cells

Terminated

Isolate immune cells from spleen and brain

with Percoll gradient

RNA isolation

0 7 9 10 11 14 15 16 18 19-21

GL261

WP1066 (o.g., 60 mg/kg)

WBRT

2 Gy x 1

Day

(C57BL/6) T cells

Other immune cells

untouched

Pan T cell MACS

Figure 2

Nanostring profiling

A

B

Spleen T cells Brain T cells Spleen T cells Brain T cells

C




B

Figure 3

C A

Co

ntro

l

WP

10

66

WB

RT

WP

10

66

+WB

RT

Dendritic Cell function




Figure 4

A

B

C

Control WBRT WP1066 WBRT+WP1066

20 uM 20 uM 20 uM 20 uM




Figure 5

G

E

*p= 0.0322

p= 0.1124

p= 0.1928

F

p= 0.5696

*p= 0.0188

p= 0.6951

*p= 0.0261

*p= 0.014

control WP1066 WBRT WP1066+WBRT

0

10

20

30

40

50

num

ber

of clu

ste

rs p

er

field

control WP1066 WBRT WP1066+WBRT

0

20

40

60

80

num

ber

of

dyads p

er

fie

ld




D

A C

B

Fold

Ch

ange

Rel

ativ

e to

Co

ntr

ol

4

3

2

1

0

Control WBRT WP1066 WP1066+ WBRT

**p=0.0018

**p=0.0096

**p=0.0037

CD45high CD11c+ CD103+ DCs

Figure 6 Research.

on November 9, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from



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