Radiation with STAT3 blockade triggers dendritic cell-T cell … · 2020-06-30 · 1 Radiation with...
Transcript of Radiation with STAT3 blockade triggers dendritic cell-T cell … · 2020-06-30 · 1 Radiation with...
1
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
2
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
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
3
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.
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
4
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.
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
5
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
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
6
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
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
7
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.
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
8
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
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
9
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
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
10
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.
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
11
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
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
12
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
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
13
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,
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
14
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
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
15
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
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
16
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+
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
17
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
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
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).
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
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
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
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
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
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
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
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.
.
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
23
REFERENCES
1. Nizamutdinov D, Stock EM, Dandashi JA, Vasquez EA, Mao Y, Dayawansa S, et al.
Prognostication of Survival Outcomes in Patients Diagnosed with Glioblastoma. World Neurosurg
2018;109:e67-e74 doi 10.1016/j.wneu.2017.09.104.
2. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al. Radiotherapy
plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005;352(10):987-
96 doi 10.1056/NEJMoa043330.
3. Tamimi AF, Juweid M. Epidemiology and Outcome of Glioblastoma. In: De Vleeschouwer S,
editor. Glioblastoma. Brisbane (AU)2017.
4. Davis ME. Glioblastoma: Overview of Disease and Treatment. Clin J Oncol Nurs 2016;20(5
Suppl):S2-8 doi 10.1188/16.CJON.S1.2-8.
5. Barker HE, Paget JT, Khan AA, Harrington KJ. The tumour microenvironment after radiotherapy:
mechanisms of resistance and recurrence. Nat Rev Cancer 2015;15(7):409-25 doi
10.1038/nrc3958.
6. Durante M, Reppingen N, Held KD. Immunologically augmented cancer treatment using modern
radiotherapy. Trends Mol Med 2013;19(9):565-82 doi 10.1016/j.molmed.2013.05.007.
7. Doucette TA, Kong LY, Yang Y, Ferguson SD, Yang J, Wei J, et al. Signal transducer and
activator of transcription 3 promotes angiogenesis and drives malignant progression in glioma.
Neuro Oncol 2012;14(9):1136-45 doi 10.1093/neuonc/nos139
nos139 [pii].
8. Hussain SF, Kong LY, Jordan J, Conrad C, Madden T, Fokt I, et al. A novel small molecule
inhibitor of signal transducers and activators of transcription 3 reverses immune tolerance in
malignant glioma patients. Cancer Res 2007;67(20):9630-6 doi 67/20/9630 [pii]
10.1158/0008-5472.CAN-07-1243.
9. Wei J, Barr J, Kong LY, Wang Y, Wu A, Sharma AK, et al. Glioblastoma cancer-initiating cells
inhibit T-cell proliferation and effector responses by the signal transducers and activators of
transcription 3 pathway. Mol Cancer Ther 2010;9(1):67-78 doi 1535-7163.MCT-09-0734 [pii]
10.1158/1535-7163.MCT-09-0734.
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
24
10. Wei J, Wang F, Kong LY, Xu S, Doucette T, Ferguson SD, et al. miR-124 inhibits STAT3
signaling to enhance T cell-mediated immune clearance of glioma. Cancer Res
2013;73(13):3913-26 doi 10.1158/0008-5472.CAN-12-4318
0008-5472.CAN-12-4318 [pii].
11. Wu A, Wei J, Kong LY, Wang Y, Priebe W, Qiao W, et al. Glioma cancer stem cells induce
immunosuppressive macrophages/microglia. Neuro Oncol 2010;12(11):1113-25 doi noq082 [pii]
10.1093/neuonc/noq082.
12. Kim JS, Kim HA, Seong MK, Seol H, Oh JS, Kim EK, et al. STAT3-survivin signaling mediates a
poor response to radiotherapy in HER2-positive breast cancers. Oncotarget 2016;7(6):7055-65
doi 10.18632/oncotarget.6855.
13. Wang Y, van Boxel-Dezaire AH, Cheon H, Yang J, Stark GR. STAT3 activation in response to IL-
6 is prolonged by the binding of IL-6 receptor to EGF receptor. Proc Natl Acad Sci U S A
2013;110(42):16975-80 doi 10.1073/pnas.1315862110.
14. Wang T, Niu G, Kortylewski M, Burdelya L, Shain K, Zhang S, et al. Regulation of the innate and
adaptive immune responses by Stat-3 signaling in tumor cells. Nat Med 2004;10(1):48-54 doi
10.1038/nm976 nm976 [pii].
15. Melillo JA, Song L, Bhagat G, Blazquez AB, Plumlee CR, Lee C, et al. Dendritic cell (DC)-specific
targeting reveals Stat3 as a negative regulator of DC function. J Immunol 2010;184(5):2638-45
doi jimmunol.0902960 [pii] 10.4049/jimmunol.0902960.
16. Lau J, Ilkhanizadeh S, Wang S, Miroshnikova YA, Salvatierra NA, Wong RA, et al. STAT3
Blockade Inhibits Radiation-Induced Malignant Progression in Glioma. Cancer Res
2015;75(20):4302-11 doi 10.1158/0008-5472.CAN-14-3331.
17. Kaushik N, Kim MJ, Kim RK, Kumar Kaushik N, Seong KM, Nam SY, et al. Low-dose radiation
decreases tumor progression via the inhibition of the JAK1/STAT3 signaling axis in breast cancer
cell lines. Sci Rep 2017;7:43361 doi 10.1038/srep43361.
18. Gao L, Li FS, Chen XH, Liu QW, Feng JB, Liu QJ, et al. Radiation induces phosphorylation of
STAT3 in a dose- and time-dependent manner. Asian Pac J Cancer Prev 2014;15(15):6161-4.
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
25
19. Pan J, Ruan W, Qin M, Long Y, Wan T, Yu K, et al. Intradermal delivery of STAT3 siRNA to treat
melanoma via dissolving microneedles. Sci Rep 2018;8(1):1117 doi 10.1038/s41598-018-19463-
2.
20. Xie TX, Huang FJ, Aldape KD, Kang SH, Liu M, Gershenwald JE, et al. Activation of stat3 in
human melanoma promotes brain metastasis. Cancer Res 2006;66(6):3188-96.
21. You S, Li R, Park D, Xie M, Sica GL, Cao Y, et al. Disruption of STAT3 by niclosamide reverses
radioresistance of human lung cancer. Mol Cancer Ther 2014;13(3):606-16 doi 10.1158/1535-
7163.MCT-13-0608.
22. Bharadwaj U, Eckols TK, Xu X, Kasembeli MM, Chen Y, Adachi M, et al. Small-molecule
inhibition of STAT3 in radioresistant head and neck squamous cell carcinoma. Oncotarget
2016;7(18):26307-30 doi 10.18632/oncotarget.8368.
23. Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al. Toll-like receptor 4-
dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat
Med 2007;13(9):1050-9 doi 10.1038/nm1622.
24. Aymeric L, Apetoh L, Ghiringhelli F, Tesniere A, Martins I, Kroemer G, et al. Tumor cell death and
ATP release prime dendritic cells and efficient anticancer immunity. Cancer Res 2010;70(3):855-
8 doi 10.1158/0008-5472.CAN-09-3566.
25. Zhang Y, Pastan I. High shed antigen levels within tumors: an additional barrier to
immunoconjugate therapy. Clin Cancer Res 2008;14(24):7981-6 doi 10.1158/1078-0432.CCR-08-
0324.
26. Madden T KR, Myer J, Culotta K, Donato N, Johansen M, et al. . The preclinical pharmacology of
WP1066, a potent small molecule inhibitor of the JAK2/STAT3 pathway. 97th Annual Meeting of
the American Association for Cancer Research 2006.
27. Iwamaru A, Szymanski S, Iwado E, Aoki H, Yokoyama T, Fokt I, et al. A novel inhibitor of the
STAT3 pathway induces apoptosis in malignant glioma cells both in vitro and in vivo. Oncogene
2007;26(17):2435-44 doi 1210031 [pii] 10.1038/sj.onc.1210031.
28. Kong LY, Wu AS, Doucette T, Wei J, Priebe W, Fuller GN, et al. Intratumoral mediated
immunosuppression is prognostic in genetically engineered murine models of glioma and
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
26
correlates to immunotherapeutic responses. Clin Cancer Res 2010;16(23):5722-33 doi 1078-
0432.CCR-10-1693 [pii 10.1158/1078-0432.CCR-10-1693.
29. Ferrajoli A, Faderl S, Van Q, Koch P, Harris D, Liu Z, et al. WP1066 disrupts Janus kinase-2 and
induces caspase-dependent apoptosis in acute myelogenous leukemia cells. Cancer Res
2007;67(23):11291-9 doi 10.1158/0008-5472.CAN-07-0593.
30. Hatiboglu MA, Kong LY, Wei J, Wang Y, McEnery KA, Fuller GN, et al. The tumor
microenvironment expression of p-STAT3 influences the efficacy of cyclophosphamide with
WP1066 in murine melanoma models. Int J Cancer 2012;131(1):8-17 doi 10.1002/ijc.26307.
31. Kong LY, Abou-Ghazal MK, Wei J, Chakraborty A, Sun W, Qiao W, et al. A novel inhibitor of
signal transducers and activators of transcription 3 activation is efficacious against established
central nervous system melanoma and inhibits regulatory T cells. Clin Cancer Res
2008;14(18):5759-68.
32. Kong LY, Wei J, Sharma AK, Barr J, Abou-Ghazal MK, Fokt I, et al. A novel phosphorylated
STAT3 inhibitor enhances T cell cytotoxicity against melanoma through inhibition of regulatory T
cells. Cancer Immunol Immunother 2009;58(7):1023-32.
33. Kupferman ME, Jayakumar A, Zhou G, Xie T, Dakak-Yazici Y, Zhao M, et al. Therapeutic
suppression of constitutive and inducible JAK\STAT activation in head and neck squamous cell
carcinoma. J Exp Ther Oncol 2009;8(2):117-27.
34. Zhou X, Ren Y, Liu A, Han L, Zhang K, Li S, et al. STAT3 inhibitor WP1066 attenuates miRNA-21
to suppress human oral squamous cell carcinoma growth in vitro and in vivo. Oncol Rep
2014;31(5):2173-80 doi 10.3892/or.2014.3114.
35. Horiguchi A, Asano T, Kuroda K, Sato A, Asakuma J, Ito K, et al. STAT3 inhibitor WP1066 as a
novel therapeutic agent for renal cell carcinoma. Br J Cancer 2010;102(11):1592-9 doi
10.1038/sj.bjc.6605691.
36. Chiu HC, Chou DL, Huang CT, Lin WH, Lien TW, Yen KJ, et al. Suppression of Stat3 activity
sensitizes gefitinib-resistant non small cell lung cancer cells. Biochem Pharmacol
2011;81(11):1263-70 doi 10.1016/j.bcp.2011.03.003.
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
27
37. Lee HT, Xue J, Chou PC, Zhou A, Yang P, Conrad CA, et al. Stat3 orchestrates interaction
between endothelial and tumor cells and inhibition of Stat3 suppresses brain metastasis of breast
cancer cells. Oncotarget 2015;6(12):10016-29 doi 10.18632/oncotarget.3540.
38. Oh T, Fakurnejad S, Sayegh ET, Clark AJ, Ivan ME, Sun MZ, et al. Immunocompetent murine
models for the study of glioblastoma immunotherapy. J Transl Med 2014;12:107 doi
10.1186/1479-5876-12-107.
39. Szatmari T, Lumniczky K, Desaknai S, Trajcevski S, Hidvegi EJ, Hamada H, et al. Detailed
characterization of the mouse glioma 261 tumor model for experimental glioblastoma therapy.
Cancer Sci 2006;97(6):546-53 doi 10.1111/j.1349-7006.2006.00208.x.
40. Hung AL, Maxwell R, Theodros D, Belcaid Z, Mathios D, Luksik AS, et al. TIGIT and PD-1 dual
checkpoint blockade enhances antitumor immunity and survival in GBM. Oncoimmunology
2018;7(8):e1466769 doi 10.1080/2162402X.2018.1466769.
41. Kim JE, Patel MA, Mangraviti A, Kim ES, Theodros D, Velarde E, et al. Combination Therapy with
Anti-PD-1, Anti-TIM-3, and Focal Radiation Results in Regression of Murine Gliomas. Clin Cancer
Res 2017;23(1):124-36 doi 10.1158/1078-0432.CCR-15-1535.
42. Wainwright DA, Chang AL, Dey M, Balyasnikova IV, Kim C, Tobias AL, et al. Durable therapeutic
efficacy utilizing combinatorial blockade against IDO, CTLA-4 and PD-L1 in mice with brain
tumors. Clin Cancer Res 2014;20(20):5290-301 doi 1078-0432.CCR-14-0514 [pii]
10.1158/1078-0432.CCR-14-0514.
43. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set
enrichment analysis: a knowledge-based approach for interpreting genome-wide expression
profiles. Proc Natl Acad Sci U S A 2005;102(43):15545-50 doi 10.1073/pnas.0506580102.
44. Dinse GE, Lagakos SW. Nonparametric estimation of lifetime and disease onset distributions
from incomplete observations. Biometrics 1982;38(4):921-32.
45. Mantel N. Evaluation of survival data and two new rank order statistics arising in its consideration.
Cancer Chemother Rep 1966;50(3):163-70.
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
28
46. Yan J, Zhao Q, Gabrusiewicz K, Kong LY, Xia X, Wang J, et al. FGL2 promotes tumor
progression in the CNS by suppressing CD103(+) dendritic cell differentiation. Nat Commun
2019;10(1):448 doi 10.1038/s41467-018-08271-x.
47. Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, et al. The immunological
synapse: a molecular machine controlling T cell activation. Science 1999;285(5425):221-7 doi
10.1126/science.285.5425.221.
48. Srivastava S, Riddell SR. Engineering CAR-T cells: Design concepts. Trends Immunol
2015;36(8):494-502 doi 10.1016/j.it.2015.06.004.
49. Austyn JM, Weinstein DE, Steinman RM. Clustering with dendritic cells precedes and is essential
for T-cell proliferation in a mitogenesis model. Immunology 1988;63(4):691-6.
50. Broz ML, Binnewies M, Boldajipour B, Nelson AE, Pollack JL, Erle DJ, et al. Dissecting the tumor
myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity.
Cancer Cell 2014;26(5):638-52 doi 10.1016/j.ccell.2014.09.007.
51. Sabins NC, Harman BC, Barone LR, Shen S, Santulli-Marotto S. Differential Expression of
Immune Checkpoint Modulators on In Vitro Primed CD4(+) and CD8(+) T Cells. Front Immunol
2016;7:221 doi 10.3389/fimmu.2016.00221.
52. Woroniecka K, Chongsathidkiet P, Rhodin K, Kemeny H, Dechant C, Farber SH, et al. T-Cell
Exhaustion Signatures Vary with Tumor Type and Are Severe in Glioblastoma. Clin Cancer Res
2018;24(17):4175-86 doi 10.1158/1078-0432.CCR-17-1846.
53. Tran Janco JM, Lamichhane P, Karyampudi L, Knutson KL. Tumor-infiltrating dendritic cells in
cancer pathogenesis. J Immunol 2015;194(7):2985-91 doi 10.4049/jimmunol.1403134.
54. Shigematsu A, Adachi Y, Koike-Kiriyama N, Suzuki Y, Iwasaki M, Koike Y, et al. Effects of low-
dose irradiation on enhancement of immunity by dendritic cells. Journal of radiation research
2007;48(1):51-5 doi 10.1269/jrr.06048.
55. Randolph GJ. Dendritic cell migration to lymph nodes: cytokines, chemokines, and lipid
mediators. Semin Immunol 2001;13(5):267-74 doi 10.1006/smim.2001.0322.
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
29
56. Yu N, Wang S, Song X, Gao L, Li W, Yu H, et al. Low-Dose Radiation Promotes Dendritic Cell
Migration and IL-12 Production via the ATM/NF-KappaB Pathway. Radiat Res 2018;189(4):409-
17 doi 10.1667/RR14840.1.
57. Ley K. The second touch hypothesis: T cell activation, homing and polarization. F1000Research
2014;3:37 doi 10.12688/f1000research.3-37.v2.
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
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
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
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
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
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 +
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
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.
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
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
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
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
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
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
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 + W B R T )
Figure 1
Control WP1066
WBRT WP1066+WBRT
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
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
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
B
Figure 3
C A
Co
ntro
l
WP
10
66
WB
RT
WP
10
66
+WB
RT
Dendritic Cell function
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
Figure 4
A
B
C
Control WBRT WP1066 WBRT+WP1066
20 uM 20 uM 20 uM 20 uM
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
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
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
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
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
Published OnlineFirst June 30, 2020.Clin Cancer Res Martina Ott, Cynthia Kassab, Anantha Marisetty, et al. efficacyinteractions in the glioma microenvironment and therapeutic Radiation with STAT3 blockade triggers dendritic cell-T cell
Updated version
10.1158/1078-0432.CCR-19-4092doi:
Access the most recent version of this article at:
Material
Supplementary
http://clincancerres.aacrjournals.org/content/suppl/2020/07/02/1078-0432.CCR-19-4092.DC1
Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://clincancerres.aacrjournals.org/content/early/2020/06/30/1078-0432.CCR-19-4092To request permission to re-use all or part of this article, use this link
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