Radiation with STAT3 blockade triggers dendritic cell-T cell interactions in the glioma microenvironment and therapeutic efficacy

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.

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.

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 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 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 37° 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 × 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. 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 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 × 22.5, with a matrix size of 256 × 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

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. 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 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 × 10 min), and rehydrated through a graded series of ethanol solutions 100% (2 times × 5 min), 90% (2 times × 5 min), 80% (1 time × 5 min), 70% (1 time × 5 min) followed by a PBS rinse (2 times × 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 × 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 × 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 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 × 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 × 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 × 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 × 5 min) and mounted with Dako fluorescence mounting medium and 22 × 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 × 10 min) and rehydrated through a graded series of ethanol solutions (100% 1 × 10 min; 95% 1 × 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 × 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 × 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 × 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, 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 × 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 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 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.

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).

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+ 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.

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 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.

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 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.

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.

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.

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 the combination. Average percentage of CD11c+ positive cells per field: control = 5.53%, WP1066 = 5.6%, WBRT = 10.6%, WBRT + WP1066 = 16.94%.

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). 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).

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.

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.

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 + 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.

DISCUSSION

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 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 administration of the combination of WP1066 and WBRT, including CD274 (PD-L1), which could also be considered in the future for clinical trial implementation.

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