key: cord-0297605-ce7d0se9
authors: Yang, Yuanfan; Brown, Michael C.; Zhang, Gao; Stevenson, Kevin; Mohme, Malte; Kornahrens, Reb; Bigner, Darell D.; Ashley, David M.; López, Giselle Y.; Gromeier, Matthias
title: Polio Virotherapy of Malignant Glioma Engages the Tumor Myeloid Infiltrate and Induces Diffuse Microglia Activation
date: 2022-05-04
journal: bioRxiv
DOI: 10.1101/2022.04.19.488771
sha: a857e06dc0e328f5e168dfdbde3ac78de04513ec
doc_id: 297605
cord_uid: ce7d0se9

Malignant gliomas commandeer abundant inflammatory infiltrates with glioma-associated macrophages and microglia (GAMM) actively promoting tumor progression. Like all cells in the mononuclear phagocytic system, macrophages and microglia constitutively express the poliovirus receptor, CD155. Besides myeloid cells, CD155 is widely upregulated ectopically in the neoplastic compartment of malignant gliomas (and solid cancers in general). Intratumor treatment with the highly attenuated rhino:poliovirus chimera, PVSRIPO, yielded long-term survival with durable radiographic responses in patients with recurrent glioblastoma (Desjardins et al. New England Journal of Medicine, 2018). Here, we studied mechanisms of PVSRIPO immunotherapy in mouse brain tumor models to decipher contributions of myeloid vs. malignant cells to antitumor efficacy. PVSRIPO treatment caused intense engagement of the GAMM infiltrate associated with substantial, but transient tumor regression. This was accompanied by diffuse microglia activation and proliferation in the normal central nervous system (CNS) surrounding the tumor, extending to the ipsilateral and even the contralateral hemispheres. PVSRIPO-instigated microglia activation occurred against a backdrop of sustained innate antiviral inflammation, associated with induction of the PD-L1 immune checkpoint on GAMM. Combining PVSRIPO with PD1/PD-L1 blockade led to durable remissions. Our work implicates GAMM as active drivers of PVSRIPO-induced antitumor inflammation and reveals profound and widespread neuroinflammatory activation of the CNS-resident myeloid compartment by PVSRIPO.

Malignant gliomas comprise a diverse and heterogeneous group of primary CNS neoplasms that respond poorly to standard of care chemoradiation. They feature dense inflammatory infiltrates, shaping a tumor microenvironment (TME) populated with GAMM [35] . Microglia-derived from the embryonic yolk sac [19] and capable of self-renewal in the CNS [2, 27] -and CNS-associated macrophages are the principal innate immune cells in the CNS [40] . Malignant gliomas actively recruit and subvert these cells, ostensibly for tumor-accommodating pro-growth, angiogenic, and immune-skewing effects (reviewed in [39] ).

Treatment of glioblastoma (GBM) with PVSRIPO achieved greater long-term survival vs. criteriamatched external controls with Overall Survival of 21% vs. 4% at 36 months [15] . Long-term survival after PVSRIPO was associated with objective radiographic responses with a median duration of >60 months. Polioviruses (PVs) use human CD155 (hCD155) as their sole host cell receptor [32] . Upon natural (oral) infection, PV targets certain lymphoid and intestinal epithelial cells and CD11c + dendritic cells (DCs)/macrophages [44] . In chimpanzees, lymphatic structures are the principal sites of PV replication [5, 41] ; widespread involvement of the lymphatic system was the defining systemic pathology of lethal PV infection in a large autopsy series mandated in a 1916 polio outbreak in the US [11] . Paralytic polio-the mechanisms of which remain obscure to this day-may be due to accidental viral targeting of anterior horn motor neurons expressing CD155 [12] , possibly a neurodevelopmental remnant [25] .

Wild type (wt) PV is highly cytotoxic with rampant viral growth in DCs/macrophages in vivo [44] and in vitro [45] . In contrast, PVSRIPO is engineered for profound attenuation [21] , evident as absent neuropathogenicity after high-dose intracerebral (I.C.) inoculation in non-human primates [16] and in human subjects [15] . In DCs/macrophages, PVSRIPO exhibits a peculiar noncytopathogenic phenotype, resulting in sublethal viral translation with lingering propagation driving sustained innate antiviral type-I interferon (IFN) dominant inflammation [9, 10, 36] .

This scenario raises questions if PVSRIPO-analogous to wt PV tropism for DCs/macrophages in vivo-targets the glioma myeloid infiltrate. Moreover, are viral effects limited to the (injected) TME or do they extend to CNS-resident myeloid cells? In this work, we used I.C. immunocompetent murine tumor models for comprehensive histology, immunohistochemistry (IHC) and

RNAseq studies of PVSRIPO immunotherapy. We report robust GAMM engagement and tumor regression, without evidence for direct viral damage to the neoplastic compartment. Beyond GAMM, we observed microglia activation immersing the tumor periphery and reaching deep into contralateral cortex. This was evident as microglia with increased cytoplasm and thickened ramified processes-highlighted on Transmembrane protein 119 (Tmem119) [4] and Ionized Ca ++ -binding adaptor protein 1 (Iba1) [1] staining-and profuse microglia proliferation upon PVSRIPO administration. RNAseq of the tumor region identified type-I/II IFN innate signaling gene signatures and upregulation of PD-L1 on GAMM. Drastic, but transient, antitumor effects of the acute neuroinflammatory response to PVSRIPO resulted in durable remissions upon combination with PD1:PD-L1 immune checkpoint blockade.

We utilized PVSRIPO produced as a good-laboratory-practice lot suitable for research purposes.

Infectious cDNA [21] was digested with MluI, in vitro transcribed with T7 polymerase (Megascript, Thermo Fisher) and full-length viral RNA was transfected into HeLa cells with DMRIE-C in Opti-MEM (both Thermo Fisher) [17] . Progeny virus recovered from transfected HeLa cell cultures was quantified by plaque assay and amplified as described previously [17] . Upon manifestation of cytopathic effects, the infected cultures were harvested, subjected to 3 freeze thaw cycles, the lysate was centrifugated at 14,000g, and the resulting supernatant was filtered through 0.1 mM syringe filters (Pall) and subjected to centrifugation through a 100-kDa cutoff spin column (Millipore). We used CT2A hCD155 and B16 hCD155 cells for I.C. implantation into C57Bl6 mice transgenic for human CD155 (hCD155-tg mice). Parental CT2A cells (a gift from Dr. P. Fecci, Duke University) and B16.F10 (ATCC) were subjected to lentiviral transduction with hCD155, followed by Fluorescence-Activated Cell sorting (FACs) with a-CD155-PE (BioLegend). Cells, confirmed mycoplasma negative (Duke Cell Culture Facility), were grown in DMEM (Invitrogen) with 10% fetal bovine serum (Sigma, #F0926) to 60-70% confluency and harvested for I.C. implantation.

Homozygous hCD155-tg mice (a gift from S. Koike, Tokyo Metropolitan Institute of Medical Science, Japan) are maintained as a breeding colony. For survival studies, 8-12 wks old, ~17-25g male and female mice were used, with roughly equal distribution in each treatment group; female mice were used for non-survival studies. Mice were housed in the Duke University Cancer Center Isolation facility under BSL2 conditions with 12-hr light/dark cycles, relative humidity of

All animal procedures were performed under a Duke IACUC-approved vertebrate animal use protocol. Briefly, prior to surgery, hCD155-tg mice were shaved on the scalp and ear-tagged.

During surgery, mice were under continuously inhaled isoflurane anesthesia and pain control (0.1 ml, 0.5% meloxicam S.C.). Mice were mounted onto stereotactic frames (Stoelting) when there was no toe pinch response; a heating pad (37 o C) was used to maintain body temperature. The scalp was sterilized (betadine + 70% ethanol), and an incision was made by a #10 blade scalpel along the sagittal suture. The surgical field was washed with 3% H2O2 to expose the bregma, which was set as the origin (x=0, y=0, z=0) of the coordinate system. A 50 μl microsyringe (Hamilton, #80901) pre-filled with tumor cells homogenously suspended in 2.4% methylcellulose was fixed onto the frame, and a keyhole was created at (x=2.0mm, z=-0.5mm) by microdrill, so the 30Gy injecting needle could pierce through the skull freely, to a depth of 3.6 mm measuring from where the bevel tip intersects with the brain surface. Each mouse received 1×10 5 CT2A hCD155 or 1×10 3 B16 hCD155 cells (5 μl), infused at 2.5 μl/min by an automatic injector. One min after completing the injection, the needle was slowly withdrawn, and the trepanation hole was sealed with bone wax (Covidien). The wound was closed with surgical glue (Vetclose, Henry Schein, Inc.) and mice were monitored during recovery until rolling over. PVSRIPO/mock treatment was performed on a day that is ~1/3 of the median survival for each tumor model (day 6 for CT2A hCD155 ; day 5 for B16 hCD155 ). Mice were divided into cohorts by a random number generator, and stereotactic intratumor infusion of PVSRIPO [5×10 7 pfu in 5 μl DMEM)/mock (5 μl DMEM)] was performed using the keyhole created for tumor implantation. Following tumor implantation, mice were weighed every 2 or 3 days until reaching the humane endpoint, defined as >15% weight loss from the highest recorded weight, or until neurological symptoms (seizure, paralysis, etc.) were evident. Mice surviving long-term (3-times median survival, >60 days) were challenged with contralateral implantation of the same number of cells.

At the sample collection interval defined by the specific study, mice were euthanized, and brains were harvested for fixation in 10% formalin (Fisher Chemical) for 1-2 days and transferred to 70% ethanol for dehydration. Brain samples were cut coronally and paraffin embedded on the cut surface for sectioning. All histology slides in this study were coronal sections (7 µm for histology; Slides were deparaffinized and subjected to IHC with primary antibodies diluted in 0.1% bovine serum albumin, followed by appropriate HRP-conjugated secondary antibodies (Supplementary Table 1 ), DAB staining, and hematoxylin and bluing agent counterstain. After staining, slides were washed in running soap water, dehydrated with a series of graded ethanol, cleaned with Sub-X, and mounted in Sub-X Mounting Medium (Leica). Manual cell counts were from three different fields selected ventral to the tumor, away from the peritumoral inflammation. IF was performed with a multiplexing approach (Visikol, Inc.). Slides were subjected to iterative rounds of staining with different antibodies (Supplementary Table 1) , interspersed with antibody-stripping procedures. Slides were scanned and assembled for visualization with high resolution to allow for co-localization analyses. The technician acquiring the data was blinded to the treatments.

All samples used to collect bulk RNA stemmed from study series 3 (Supplementary Table 3 

Survival data were analyzed by Kaplan Meier curves; when comparing two groups significance was determined by Log Rank test. For comparing histology measurements between treatment and control groups student t test with unequal variance was used; Cohen's effect size of >0.8 was defined as a 'large effect'. Other assay-specific statistical tests are indicated in the corresponding figure legends. P values of <0.05 were considered significant. All statistical analyses were performed using Prism 9 software version 9.0; error bars represent Standard Error of the Mean (SEM). All data points reflect individual specimens, independent experimental repeats, or mice.

CT2A is a 20-methylcholanthrene-induced murine anaplastic astrocytoma [43] that faithfully recapitulates key properties of human malignant glioma [30] . CT2A tumors have a high take rate, grow aggressively, and are resistant to immune checkpoint blockade [38] . Mouse CD155 does not function as a PV receptor; therefore, to recapitulate the clinical scenario, we transduced cells with hCD155 (CT2A hCD155 ) for implantation into hCD155-tg mice. To optimize I.C. mouse tumor models for histopathological analyses of all brains in all cohorts, we adjusted multiple variables to ensure consistent tumor growth contained within the right hemisphere. This included needle size/use of a drill for trepanation, inoculum (number of cells), and inoculation depth for maximal tumor take with minimal incidence of injection tract infiltration/extra-parenchymal growth. With these measures, we achieved 100% tumor take with 93.3% of tumors contained within the injected hemisphere per gross pathological exam (Supplementary Table 2 ).

Six days after I.C. CT2A hCD155 implantation in hCD155-tg mice, a single stereotactic infusion of PVSRIPO (5×10 7 pfu) or mock (DMEM) was done (Fig. 1) . This interval was chosen because histology of brains harboring CT2A hCD155 tumors 6 days post implantation revealed sizeable, established striatal growths (Fig. 1) . In three independent executions of this experiment, tumorbearing brains were harvested from mice euthanized at baseline (day 6), and from mice in the post PVSRIPO/mock cohorts collected at days 8, 10, 12, 15 or 18 (Fig. 1) . Brains of all mice (n=55) enrolled in the 3 series were collected for histology (Supplementary Table 3 ). Because some mice reached humane end points (>15% weight loss, clinical symptoms) on/before day 18, the systematic part of this study (n=49) was not extended beyond day 15, to avoid attrition bias.

Coronal FFPE sections of the tumor implantation site-using the needle track and anterior commissure as landmarks-were H&E stained for post-therapy histology in comparison with baseline and time-matched mock controls. Sample micrographs were assembled into a composite image to illustrate the treatment response of CT2A CD155 tumors to PVSRIPO over time ( Fig. 1) . This revealed a dramatic, but transient, antitumor effect evident as distinct histological appearance and reduced tumor size at day 8, followed by tumor relapse by day 15 (Fig. 1) . At baseline (day 6), the average cross-sectional area of tumor was 1.2 mm 2 , which expanded by day Albeit rare, this is notable because CT2A glioma disseminates widely [30] .

To evaluate the PVSRIPO treatment effects noted in H&E-stained whole brain sections (Fig. 1) , Table 3 were de-identified and submitted for blinded review by a Board-certified neuropathologist (G.Y.L.) to assess nine specific histological features (Fig. 2) . This analysis revealed key features distinguishing PVSRIPO treated brains from the mock controls: (i) marked increase in microglia; (ii) discohesiveness throughout tumor; (iii) discohesiveness at periphery of tumor; and (iv) perivascular inflammation (Fig. 2a, b) .

The predominant histological feature of the PVSRIPO response, present in all samples, was marked increase in microglia (Fig. 2) . Microglia were evaluated in peritumoral brain parenchyma.

Microglial activation was defined as aggregates of rod-shaped nuclei located immediately adjacent to the tumor. To meet criteria for an increased density of activated microglia, exclusion of other key cell types was required. The cells with rod-shaped nuclei had to lack visible cytoplasm.

Abundant eosinophilic cytoplasm with ramified processes would support activated astrocytes, while abundant foamy cytoplasm would raise a differential of bone-marrow derived macrophages. Neither feature was present in regions identified as having activated microglia. While a mild increase in microglia (located mostly along the needle track) was found in mock-treated tumors relative to contralateral normal brain, we observed marked enrichment of activated microglia in the tumor periphery and throughout the brain after PVSRIPO treatment (see below).

Histopathological evidence for a microglial response to PVSRIPO (Fig. 2) requires rigorous determination of cell identity. Glioma implantation and virus inoculation induce CNS peripheral monocytic infiltrates which must be distinguished from tissue-resident microglia. Since Iba1 is also induced in activated CNS-associated macrophages or infiltrating peripheral monocytes [13] , we added IHC for transmembrane protein 119 (Tmem119), which specifically marks microglia in the CNS [4] , eg. in the context of RNA virus CNS infection [13] .

To eliminate the tumor as a source for Iba1 + monocytic infiltration, we assessed microglia in nontumor-bearing brains treated with PVSRIPO vs. mock (Fig. 3) . IHC of Iba1 and Tmem119 demonstrated diffuse microglial activation, characterized as increased cytoplasm and thickened ramified processes, throughout the PVSRIPO-treated hemisphere (Fig. 3a) . In mock-treated brains, Iba1 + cells occurred at the injection site only; these cells likely represent infiltrating monocytes, as they did not stain for Tmem119 in PVSRIPO/mock treated mice (Fig. 3a) . CNS-wide distribution of Iba1 + /Tmem119 + cells in PVSRIPO treated brains, excluding the injection site (Fig.   3a) , provides definitive evidence for diffuse microglia activation by PVSRIPO. Higher magnification images of Iba1 (Fig. 3b) and Tmem119 (Fig. 3c) staining in cortex (distant from the injection site) revealed Tmem119 + cells with morphologic changes characteristic of activated microglia only in PVSRIPO treated brains (Fig. 3c) .

To corroborate our findings in CT2A hCD155 murine glioma, we selected the I.C. B16.F10 (melanoma) model transduced with hCD155 [10] . B16 is spontaneous and one of the least intrinsically immunogenic mouse tumor models [28] . PVSRIPO/mock treatment of I.C. B16 hCD155 -harboring mice was done on day 5 (instead of day 6 with CT2A hCD155 ) and brains were harvested on day 7 (instead of day 8 with CT2A hCD155 ) due to shorter median survival (~14.5 days for B16 hCD155 vs.

~19.5 days for CT2A hCD155 ). Blinded pathological review of mock (n=2) vs. PVSRIPO-treated (n=3) B16 hCD155 -bearing brains (study day 7), revealed similar treatment effects: mock-treated tumors had clearly delineated borders with minimal inflammation, whereas PVSRIPO-treated tumors were smaller, discohesive, admixed with inflammation and markedly increased GAMM in and around the tumor with prominent peritumoral and perivascular inflammation ( Supplementary  Fig. 2a, b) . The day 8 post-PVSRIPO antitumor effect noted in CT2A hCD155 was evident in Ki67 IHC (Supplementary Fig. 2c ) and CD68 IHC signal was profusely enhanced in the tumor bed and surrounding brain (Supplementary Fig. 2d) .

Having confirmed the microglial nature of Iba1 + cells throughout the ipsilateral hemisphere by presence of both highly ramified morphology and Tmem119 positivity (Fig. 3) , we selected representative slides from PVSRIPO/mock-treated CT2A hCD155 glioma-bearing mice collected on day 8 (n=2 mock/4 PVSRIPO) for multiplex IF staining of Iba1, Ki67, PD-L1 (Fig. 4a, b) and confirmatory IHC of Iba1 (Fig. 4c, d) . We focused on three regions: a central portion of the tumor, ipsilateral tumor-adjacent normal brain close to the anterior commissure ( Fig. 4a-d) , and distant contralateral cortex (Fig. 4c, d) . Iba1 staining delineated tissue-resident microglia with characteristic ramified processes that emanate from a cell body in peritumoral normal brain ( Fig.   4a-d) . In PVSRIPO-treated brains, the presence of activated microglia was pervasive, with prominent microglia activation, both in peritumoral normal brain and in contralateral cortex (Fig.   4b, d) . Profuse microglia activation was evident in all virus-treated brains, but none of the mocktreated controls. We confirmed microglia identity by staining with Tmem119 in adjacent sections (data not shown). Further signs of engaged microglia in PVSRIPO-treated brains were evident upon multiplex staining with Iba1 and Ki67 (Fig. 4a, b) . In mock-treated animals, ipsilateral normal brain was devoid of Ki67 staining (Fig. 4a) , while in PVSRIPO-treated animals Iba1 + cells with highly ramified processes co-stained with Ki67 (Fig. 4b) , supporting widespread active microglia proliferation in brain parenchyma.

Beyond microglia activation in ipsilateral/contralateral brain, PVSRIPO induced profound proinflammatory engagement in the TME. In mock-treated animals, PD-L1 staining in the tumor proper was sparse (Fig. 4a) . In contrast, abundant PD-L1 co-staining with Iba1 in the tumor bed in PVSRIPO-treated mice indicated type-I IFN mediated proinflammatory activation of GAMM (PD-L1 is an IFN-stimulated gene; PVSRIPO induces PD-L1 in human DCs in vitro [36] ; Fig. 4b ).

Iba1 + cell activation extending to normal ipsilateral brain and the contralateral hemisphere ( Supplementary Fig. 3b) , and active microglia proliferation in normal brain parenchyma ( Supplementary Fig. 3f) were also observed in all cases of the I.C. B16 hCD155 model treated with PVSRIPO that were submitted for multiplex IF analysis (n=3).

To further document the PVSRIPO treatment effect, we co-stained sections from the brains of CT2A hCD155 -bearing mice after PVSRIPO/mock treatment by multiplex IF staining of CD45, PD-L1 and Ki67 (Fig. 5) . In mock-treated animals, the CD45 + compartment remained well organized, interwoven with and mostly restricted to the neoplastic lesion itself (Fig. 5a, b) with scarce and punctate PD-L1 expression (Fig. 5c) . Accordingly, Ki67 staining was intense in the neoplastic lesion, with some scattered signal in the peritumoral periphery (Fig. 5d) . In PVSRIPO-treated animals, however, CD45 staining was no longer confined to the malignant lesion, but extended far into brain parenchyma, forming a vast, undulating inflammatory reaction that engulfed much of the tumor-bearing hemisphere (Fig. 5e, f) , replete with PD-L1 signal (Fig. 5g) . Ki67 + cells were greatly diminished in the tumor itself (in accordance with reduced tumor size; Fig. 2c ) and were present throughout the hemisphere (Fig. 5h) . In PVSRIPO-treated animals, Ki67 + cells located outside the tumor region largely co-stained with Iba1 and demonstrated highly ramified processes, suggesting proliferating microglia ( Fig. 5f; Supplementary Fig. 3f) ; or, Ki67 + cells costained with CD3 in a tight perinuclear pattern, suggesting proliferating T cells ( Supplementary   Fig. 5b) . Calculation of the density of CD45 + cells in CT2A hCD155 tumor-adjacent normal brain demonstrated that PVSRIPO treatment induced an ~10-fold influx of such cells into peritumoral parenchyma; the same effect was observed in the B16 hCD155 model (Supplementary Fig. 4a) . 

Co-staining with CD3 suggested that some of the Ki67 + cells in PVSRIPO/mock-treated CT2Abearing brains were T lymphocytes (Supplementary Fig. 5) . However, in contrast to mock-treated mice, where Ki67 + CD3 + cells occurred mostly within the tumor bed (Supplementary Fig. 5a) , such cells were disseminated throughout the peritumoral circumference of PVSRIPO-treated CT2A hCD155 (Supplementary Fig. 5b) and B16 hCD155 -bearing mice (Supplementary Fig. 6 ). In aggregate, our IF and IHC studies revealed a broad range of proinflammatory effects engulfing the CNS after intratumor PVSRIPO treatment. The effects were noted for their quality, eg. profuse microglia proliferation in the normal CNS and robust PD-L1 induction on GAMM and in the tumorbearing hemisphere; and their extent, eg. diffuse microglia activation throughout the brain.

To study PVSRIPO-induced transcriptomic landscape alterations, we selected 20 CT2A hCD155 Table 3 ) collected at different intervals pre-and post PVSRIPO/mock infusion. Tumor region RNA was recovered from FFPE tissue sections for bulk RNA sequencing. Differential expression analysis revealed PVSRIPO-induced up-regulation of 380 and downregulation of 10 genes (applying a log2-fold change cut-off of 2.0 and p-value cut-off of 0.05) compared to mock controls at day 8 (Fig. 6a) . This ratio dropped to 18 and 7 genes, respectively, at day 12 (Fig. 6a) . Consistent with the antitumor and inflammatory effects observed, PVSRIPO induced a profound but transient transcriptional response that peaked 48h post treatment. Gene set enrichment analyses (GSEA) interrogating Hallmark Genesets [29] revealed several recurring genesets in PVSRIPO-treated brains, led by 'IFNa response' and 'IFNg response'. Meanwhile, cell cycle progression genesets ('Myc targets V1/2', 'G2M checkpoint', 'E2F targets') were downregulated (Fig. 6b) . These results are consistent with the histological evidence for tumor regression and a broad, macrophage/microglia-centered innate antiviral inflammatory reaction.

RNAseq also provided an opportunity to assess viral RNA (vRNA) counts in the TME (Fig. 6c) .

There were no reads mapping to vRNA at baseline, prior to PVSRIPO infusion (day 6) or in any mock-treated samples (Fig. 6c) . At day 8, 48h post PVSRIPO infusion, we detected 15.9-per million input vRNA reads. After day 8, the estimated viral load quickly diminished over time (Fig. 6c) , in agreement with prior data in mouse tumor models treated with PVSRIPO [8, 10, 23] . Finally, we employed the mouse version of the Microenvironment Cell Populations (MCP)-counter algorithm [3] to calculate the abundance of tissue-infiltrating immune and stromal cells in the TME over time (Fig. 6d) . PVSRIPO induced tumor infiltration of myeloid-derived innate immune cells at day 8 (Fig. 6d) . This was followed by infiltration of CD8 + T cells and memory B cells on day 12 (Fig. 6d) .

The total amount of immune/stromal infiltrates diminished over time, in step with progressive resolution of the acute inflammatory response elicited by PVSRIPO therapy (Fig. 6d) .

Robust type-I IFN-dominant antiviral inflammation with widespread PD-L1 induction against the backdrop of a profoundly engaged myeloid infiltrate, suggests that PVSRIPO's antitumor effects could be buttressed with PD1:PD-L1 immune checkpoint blockade. Thus, we assessed antitumor efficacy of PVSRIPO combined with a-PD-L1 (Fig. 7) or a-PD1 (Supplementary Fig. 7a) . To mimic the clinical scenario with pre-existing anti-PV immunity and a PV vaccine boost given prior to PVSRIPO infusion [15], mice were immunized against PV 47 and 32 days prior to CT2A hCD155 implantation (Fig. 7) . CT2A hCD155 tumor-bearing mice were treated with mock/PVSRIPO as shown ( Fig. 1) , followed by a-PD-L1 or isotype-matched control antibodies (Fig. 7) . Combining PVSRIPO with a-PD-L1 had a minor effect on median survival (24 days vs. 19 days for the mock + IgG group; Fig. 7a) , but a significant effect on overall survival (OS) with 36% complete remission at day 60 ( Fig. 7a) . PVSRIPO + a-PD-L1 therapy-induced remission was resistant to contralateral rechallenge with CT2A (Fig. 7a) , which was lethal in 6 age-matched, treatment-naïve control mice.

The extension of OS with PVSRIPO + a-PD-L1 was eliminated upon CD8 + T cell depletion (Fig. 7b) .

To assess the role of PVSRIPO-induced GAMM engagement in immunotherapy efficacy of PVSRIPO + a-PD-L1 and of a-PD-L1 alone, treatment was repeated in animals treated with clodronate liposome-mediated macrophage depletion (Supplementary Fig. 7b) . Incomplete GAMM depletion (~30% reduction of GAMM per CD68 + staining) decreased the combination therapy effect from 40.0% to 16.7% (Supplementary Fig. 7b ).

From their inception [31] , viral anti-glioma strategies were predicated on a concept of viral infection and cytopathogenicity in neoplastic cells, based on robust virus growth in high-passage in vitro tumor models. In this context, CNS resident innate immune cells often were deemed an obstacle to therapy due to their role in clearing viral agents administered intratumorally [18] .

PVSRIPO, by virtue of widespread CD155 upregulation in malignancy [24] , including malignant glioma [12, 33] , infects and damages cancerous cells via antiviral IFN responses [46] and cytotoxic viral proteins in vitro [6, 7] . Yet, studies of PVSRIPO in ex vivo GBM slices, a clinically relevant model with authentic cellular heterogeneity, show that neoplastic cells are not the main target for its biological activities [10] . Our studies in I.C. mouse tumor models confirm that PVSRIPO mainly provokes responses in the GAMM compartment in vivo, unleashing type-I IFN dominant engagement of the notoriously immunosuppressive glioma myeloid infiltrate. This reverberates with PV's natural cell type-specificity for migratory/lymphoid DCs and macrophages [44] .

PVSRIPO therapy of I.C. mouse tumor models caused a rapid decrease of tumor size evident as reduced Ki67 + cells in the tumor bed, discohesive architecture, and a decline in cell cycle-and proliferation-related gene expression. Absent vRNA accumulation in the tumor all but excludes the possibility of direct viral cell killing to account for this. This is consistent with PVSRIPO therapy of hCD155-tg hosts (implying PVSRIPO targeting of myeloid cells) occurring with hCD155-negative tumor implants that are resistant to infection [10] . Indeed, CD8 + T cell depletion and clodronate liposome studies indicate that PVSRIPO-instigated antitumor effects are immune mediated. Yet, we cannot rule out a role for viral infection of-and cytopathogenic damage to malignant cells in the antitumor effect, even if it is sub-lethal and non-productive.

The main histological finding after PVSRIPO was marked microglia activation engulfing virtually the entire brain. Microglia are the bulwark of the CNS innate antiviral response [14] . 'Classic' viral encephalitis, eg. the syndromes modeled in mice infected with rhabdo-(vesicular stomatitis virus) [13] , flavi-(eg. West Nile virus) [42] , or neurotropic coronaviruses [26] , share common features implicated in triggering microglia activation [14] . They each infect neurons and inflict substantial neuronal damage; they infect and damage astro-and/or oligodendrocytes; they induce peripheral monocytic infiltration in the CNS. In viral encephalitis models, microglia activation occurs in close proximity to these triggers, eg. foci with viral propagation in-and damage to CNS cells [37] . PVSRIPO CNS infection lacks all of these triggers. PVSRIPO is devoid of cytopathogenic potential in neurons [34] ; it is, thus, non-neuropathogenic in human subjects [15] , non-human primates [16] , and hCD155-tg mice [22] . PVs do not infect astro-/oligodendrocytes. I.C. PVSRIPO did not induce peripheral monocyte infiltration beyond the needle track.

Unambiguously identifying microglia in the virus-infected CNS is difficult, especially against the backdrop of peripheral monocytic infiltration [14] . Tmem119 staining of cells with morphological features of activated microglia defines this compartment to respond to PVSRIPO. There was profuse proliferation of such cells, evident as Ki67 + co-staining, in the brain. Microglia activation and proliferation induced by PVSRIPO, unlike classic viral encephalitis, was diffuse and extensive, consistent with the absence of focal viral cytopathogenicity. Rather, this pattern of microglia activation may reflect PVSRIPO's non-lethal phenotype in cells of the mononuclear phagocytic system yielding sustained type-I IFN dominant inflammation [9, 10, 36] . Accordingly, the prevailing transcriptional signatures emerging from RNAseq analyses of the tumor region post PVSRIPO were antiviral inflammatory in nature, including upregulation of PD-L1.

The dramatic early treatment effects of PVSRIPO were transient and, in ~90% of cases, gave rise to lethal tumor progression. Our studies suggest that turning acute inflammatory antiviral reactions into durable remission may be achieved by combining PVSRIPO with immunomodulatory agents capable of blocking natural resistance points of the antiviral immune response, eg. with anti-PD-L1 or PD1 immune checkpoint blockade.

Our investigations demonstrate that, rather than a detriment to cancer virotherapy, the GAMM infiltrate is a promising substrate for viral immune stimulatory actions. Beyond proinflammatory engagement of the tumor myeloid infiltrate, the quality, depth, and anatomical reach of PVSRIPO-induced microglia activation may be indispensable for generating functional antitumor CD8 + T cell immunity in malignant gliomas, notorious immune deserts intrinsically resistant to systemic immunotherapy approaches [20] . 

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Expression of the human poliovirus receptor/CD155 gene during development of the central nervous system: implications for the pathogenesis of poliomyelitis

MAC2 is a long-lasting marker of peripheral cell infiltrates into the mouse CNS after bone marrow transplantation and coronavirus infection

Repopulated microglia are solely derived from the proliferation of residual microglia after acute depletion

Immunogenicity of murine solid tumor models as a defining feature of in vivo behavior and response to immunotherapy

The Molecular Signatures Database (MSigDB) hallmark gene set collection

Standardization of an orthotopic mouse brain tumor model following transplantation of CT-2A astrocytoma cells

Experimental therapy of human glioma by means of a genetically engineered virus mutant

Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily

Poliovirus receptor CD155-targeted oncolysis of glioma

Cell-type-specific repression of internal ribosome entry site activity by double-stranded RNA-binding protein 76

Macrophages in experimental and human brain tumors. Part 2: studies of the macrophage content of human brain tumors

Genetically stable poliovirus vectors activate dendritic cells and prime antitumor CD8 T cell immunity

T cell engagement of crosspresenting microglia protects the brain from a nasal virus infection

Immunocompetent murine models for the study of glioblastoma immunotherapy

Glioblastoma-associated microglia and macrophages: targets for therapies to improve prognosis

Innate immunity in the central nervous system

Pathogenesis of poliomyelitis; reappraisal in the light of new data

TLR signaling controls lethal encephalitis in WNV-infected brain

Ganglioside distribution in murine neural tumors

Pathogenic Events in a Nonhuman Primate Model of Oral Poliovirus Infection Leading to Paralytic Poliomyelitis

Dendritic cells and macrophages are productively infected by poliovirus

Engineered Oncolytic Poliovirus PVSRIPO Subverts MDA5-Dependent Innate Immune Responses in Cancer Cells