Functionality of PTENα expressed by HSV-P10

To evaluate the functionality of PTENα expressed by HSV-P10, we tested the impact of its infection on the PI3K/AKT signaling pathway³⁹. Western blot analysis of infected cells revealed increased phosphorylation of AKT upon infection with control HSVQ virus in all breast and glioma tumor cell lines tested, while HSV-P10 infected cells revealed reduced phosphorylated AKT compared with control virus infection (Fig. 2a). Apart from functioning as an intracellular lipid phosphatase that can regulate PI3K signaling, PTENα has been shown to be secreted and then re-enter adjacent cells to inhibit PI3K signaling³⁹ and also to activate the mitochondrial electron transport chain through hyper-activation of cytochrome C, resulting in increased mitochondrial membrane potential and ATP production⁴⁰. HSV-P10 infection did result in a small amount of secreted PTENα that could be detected in concentrated (20×) conditioned medium of infected U87EGFR cells (Fig. 2b). Additionally, immunofluorescent staining of pAKT in Met1 and DB7 cells infected with HSVQ or HSV-P10 revealed that while HSVQ infection increased pAKT staining within GFP positive cells, HSV-P10 infection did not (Supplementary Figure ¹). Spectral quantification of immunofluorescent images across a straight line encompassing two cell widths of two representative adjacent cells, confirmed an increase in pAKT (red) staining within GFP positive (green) HSVQ-infected cells compared with neighboring GFP negative cells (Fig. 2d middle panel). HSV-P10 infection did not reveal infection associated induction of pAKT (Fig. 2d bottom panel). Similar results were observed in the DB7 murine breast cancer cells (Fig. 2d). Next we tested the impact of HSV-P10 on mitochondrial membrane potential using membrane-permeant JC-1 dye. This dye shows a potential-dependent mitochondrial accumulation resulting in aggregates that display a fluorescence emission shift from green (~529nm) to red (~590nm). Consistent with the known function of PTENα, we observed an increase in mitochondrial membrane potential in three (LN229, U87ΔEGFR, and DB7) out of four cell lines infected with HSV-P10 relative to control HSVQ-infected cells (Fig. 3a). U251T3 glioma cells possessed a high mitochondrial membrane potential prior to infection, and did not show a further change after HSV-P10 infection. The HSV-P10 induced change in mitochondrial membrane potential positively affected overall ATP production observed in both secreted conditioned medium (Fig.​(Fig.3b)3b) and cell lysates (Fig. 3c). To evaluate the significance of HSV-P10 mediated inhibition of AKT phosphorylation on increased ATP production we repeated the above experiment in cells treated with and without LY294002 a PI-3 Kinase inhibitor. Western blot analysis confirmed the reduction of pAKT in LY294002 treated infected and uninfected cells (Fig. 3d). While HSV-P10 treatment increased ATP production over HSVQ, treatment with LY294002 did not increase ATP release (Fig. 3e). Collectively these results show that HSV-P10 expressed PTENα, retained both mitochondrial targeting and PI3K inhibitory functions of PTEN, and that the ATP release mediated by HSV-P10 was independent of its effect on AKT phosphorylation.

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Fig. 2

HSV-P10 AKT inhibition. a Western blots of lysates from the indicated cells infected with HSVQ (Q) or HSV-P10 (P) or left untreated (NV). Infected cells (MOI=1) were lysed 24hpi and probed for expression of PTENα, pAKT (S473), Akt, and GAPDH. L indicates the moleculcar weight ladder. b Western blot for secreted PTEN expression in conditioned medium. Culture media was concentrated 20X from U87EGFR cells infected 24h with either HSVQ (Q) or HSV-P10 (P10) and probed for secreted PTENα. L indicates the molecular weight ladder. c Flow cytometric analysis of pAkt-S473 and virus encoded GFP in infected U87EGFR cells 15hpi. d Spectral analysis of fluorescent intensity along a random line drawn across the view field of one or two cells visualizing pAKT (red), DAPI (blue) and GFP (green) of the indicated cells. y-axis represents arbitrary fluorescence units (AFU). Note the higher intensity of pAKT in HSVQ-infected (GFP positive) cells relative to adjacent uninfected (GFP negative) cell (middle panel). Note the lack of increased pAKT intensity in HSV-P10 infected (GFP positive) cells relative to adjacent uninfected (GFP negative) cells (bottom panel)

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Fig. 3

Effects of PTENα on HSV-P10 infected cells. a Increased mitochondrial membrane potential in HSV-P10 infected cells 6hpi. Seeded cells were infected with either HSVQ or HSV-P10 at MOI=0.5 for 6h, and mitochondrial membrane potential indicated by JC-1 red fluorescence was imaged using fluorescence microscopy. Scale bar=100µm. b ATP release from infected cells 24hpi. Data shown are mean ATP concentration in conditioned media from infected cells 24hpi±S.D. Statistical analysis performed with a 2-tailed Student’s T-test. (n=3/group, **p<0.01, ***p<0.001). c ATP measured in cell lysates of infected cells 24hpi. Data shown are mean ATP concentration 24hpi±S.D. Statistical analysis performed with a 2-tailed Student’s T-test. (n=3/group, **p<0.01, ***p<0.001). d Western blot of DB7 cells 24hpi with HSVQ (Q), HSV-P10 (P), or left untreated (NV)±PI3Ki (LY294002). L indicates the molecular weight ladder. e ATP release from DB7 or MDA-468 cells 12hpi with HSVQ or HSV-P10. HSVQ and PI3Ki treated samples were also evaluated for ATP release. Data shown are mean ATP concentrations in conditioned media from infected cells±SEM. Statistical analysis performed with a 2-tailed Student’s T-test (n=6/group, *p<0.05, ***p<0.001, n.s.=not significant)

PTENα does not inhibit HSV-P10 kinetics

To test whether the expression of PTENα affected viral replication and/or tumor cell killing, we infected the indicated tumor cells with either control HSVQ (blue) or HSV-P10 (red) and monitored virus infection by quantifying GFP expression in infected cells by real time live cell fluorescent imaging using a Cytation 5 Cell Imaging Multi-Mode Reader in conjunction with a BioSpa 8 Automated Incubator (BioTek Instruments, INC.) over time (Fig. 4a). While HSV-P10 appeared to have superior kinetics of virus replication (Fig. 4a), it did not appear to correlate with PTEN and or activated base line AKT status of these cells in this small sample size (Supplementary ¹). The faster kinetics implied that cells infected with HSV-P10 would burst faster than control HSVQ-infected cells. Consistent with this, tumor cells were more sensitive to HSV-P10 induced killing at lower MOIs than control HSVQ-infected cells (Fig. 4b). Next we measured virus yield from cultures infected with either HSV-P10 or control HSVQ. While HSV-P10 did appear to improve the kinetics of virus replication in infected cells (Fig. 4a), it did not appear to significantly affect the burst size or total virus yield in (Fig. 4c). Consistent with a similar burst size of the viruses, the killing of tumor cells at higher MOIs, when a significant portion of the culture is infected, was found to be similar for both the viruses (Fig. 4d).

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Fig. 4

Kinetics of HSV-P10. a Comparison of HSVQ and HSV-P10 virus replication in the indicated cells. Seeded tumor cells were infected at the designated MOIs and GFP expression was monitored for 24–48h utilizing the Cytation 5 Cell Imaging Multi-Mode Reader in conjunction with a BioSpa 8 Automated Incubator (BioTek Instruments, INC.). GFP object count was quantified and graphed as an average of 4 wells per treatment group±SEM. Black line: no virus, blue line: HSVQ, red line: HSV-P10. b Cytolytic activity of HSVQ (blue line) vs. HSV-P10 (red line) at a low MOI. Cell viability of HSVQ or HSV-P10 infected tumor cell lines at MOI=0.0625 for all cells at the indicated time points after infection as measured by MTT. Data shown are percentage of viable cells relative to uninfected controls±S.D. (n=6). c Comparison of HSVQ and HSV-P10 viral yield (burst size). Data shown are median titers from cultures±S.D. (n=3/group). d Cytolytic activity of HSVQ (blue line) vs. HSV-P10 (red line) at high MOI. Cell viability of HSVQ or HSV-P10 infected tumor cell lines at MOI=0.5 for all cells at the indicated time points after infection as measured by MTT. Data shown are percentage of viable cells relative to uninfected controls±S.D. (n=6)

To evaluate the safety of an oHSV encoding for PTENα, we compared the infection and toxicity of HSVQ and HSV-P10 towards normal HUVEC and human neuronal cells. Briefly the spread of viral infection in normal HUVEC and U251T3 glioma cells was monitored over time relative to cancer cells by quantifying GFP expression in infected cells by real time live cell fluorescent imaging. While both HSVQ and HSV-P10 showed evidence of rapid virus spread through the culture in U251 cancer cells, this was highly attenuated in primary normal HUVEC cell cultures (Fig. 5a, b). Primary neuronal cultures differentiated from iPSCs derived neuron stem cells were cultured in matrigel-coated plates in neurobasal media containing BDNF (10ng/ml, Peprotech) and GDNF (10ng/ml, Peprotech) for 4 weeks. The differentiation of cells to neurons was analyzed by immunofluorescence staining for microtubule-associated protein 2 (MAP2) and neuron-specific beta-III tubulin (Tuj-1) (Fig. 5c). The differentiated neurons and MDA-468 cells were infected with HSVQ or HSV-P10 with MOI 0.1 or MOI 0.5 for 72h. As expected both HSVQ and HSVP-10 could infect primary human neurons, and consistent with increased HSV-P10 spread in cancer cell cultures, a greater number of MDA-MB-468 cells were positive for GFP at either MOI, with HSV-P10 relative to HSVQ (Fig. 5d). The apoptosis of infected neurons and MDA-468 cells was analyzed by Annexin-V/PI staining of HSV-infected neurons (Gating on MAP2+HSV-GFP+cells, Supplementary Figure ²). Consistent with the known clinical safety of HSV viruses deleted for viral neurovirullence genes ICP34.5, we observed no evidence of increased apoptosis towards these cells in vitro (Fig. 5e). This suggests that PTEN expression by the virus did not alter the safety profile of the virus in normal cells (Fig. 5d, e). MDA-MB-468 tumor cells were used as controls for the toxicity assay and showed increased sensitivity to HSV-P10 relative to HSVQ.

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Fig. 5

Safety of HSV-P10. Viral spread of HSVQ (blue line) and HSV-P10 (red line) in cultures of HUVEC (a) and U251T3 tumor cells (b) was assessed using the Cytation 5 live imaging system, where GFP expression was monitored over time. GFP object count was quantified and graphed as an average of 4 wells per treatment group±SEM. c Immunofluorescent staining of differentiated neurons showing MAP2 and Tuj1 staining. Scale bar=50µm. d Infection (GFP) of neurons and MDA468 cells followed by GFP visualization. Data shown are histograms of the indicated cells infected with HSVQ or HSV-P10. e Cytolytic activity (PI/Annexin-V) in neurons was assessed by flow cytometry 24hpi. Dot blots of annexin and PI staining of infected (GFP positive) neurons and MDA-468 cells

HSV-P10 offers a therapeutic benefit and antitumor immunity

Next, we tested the impact of HSV-P10 on antitumor efficacy, in a murine model of breast cancer brain metastases (Fig. 6b) and intracranial human GBM tumors in nude mice (Supplementary Fig. ³). To evaluate the impact of PTENα on antitumor efficacy, we treated tumor-bearing mice with a single dose of HSVQ or HSVP-10 (Fig. 6a, Supplementary Figure ^3a). We observed that while HSVQ treated mice showed an improvement in survival compared to saline control mice, HSV-P10 treatment provided an even greater improvement in overall survival compared to HSVQ treatment (Fig. 6b). Approximately 42% (16 out of 38) of mice treated with HSV-P10 were long-term survivors, showing no indication of tumor burden 90 days post tumor implantation (by MRI). Complete response was confirmed in these mice by MRI imaging (day>90 after tumor implant), which revealed an absence of visible tumor in the brains of HSV-P10 mice that survived greater than 90 days after tumor implant (Fig. 6c). The presence of long-term survivors after treatment with HSV-P10 in an immune-competent animal model indicated the engagement of the host antitumor immune response in animals treated with HSV-P10. To test the impact of HSV-P10 on antitumor immunity, we assessed the ability of mice treated with HSV-P10 to develop an antitumor immune memory response. MRI confirmed complete responders (survival greater than 90 days) treated with HSV-P10 and tumor-naïve age-matched control mice, were inoculated with 100,000 DB7 cells in the left (contralateral) brain hemisphere. While 100% of the tumor-naive age-matched control mice succumbed to tumor burden, all of the HSV-P10 treated mice rejected the rechallenge and remained tumor free (Fig. 6d). Complete tumor rejection was confirmed by MRI on day 45 following second tumor rechallenge (Fig. 6e).

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Fig. 6

HSV-P10 enhances overall survival of mice bearing breast cancer brain metastases. a Schematic representation of animal studies. b Survival curve of DB7 brain tumor-bearing FVB/N mice treated intratumorally with saline control (blue line: n=41), HSVQ (red line: n=38), or HSV-P10 (green line: n=38) 7 days post tumor cell implantation. Significance in survival was assessed by Logrank (Mantel–Cox) test comparing only two survival curves per test (**p<0.01, ****p<0.0001). c Brain magnetic resonance imaging (MRI) of one representative long-term survivor mouse treated with HSV-P10 from Fig. 5b>90 days post tumor cell implantation. White arrows indicate the needle track of the initial injection site. d Survival curve of HSV-P10 treated long-term survivor mice (green line: n=5) from Fig. 5b contralaterally inoculated with 100,000 DB7 tumor cells vs. naive age-matched control mice (blue line: n=8). Significance in survival was assessed by Logrank (Mantel–Cox) test (***p<0.001). e Brain MRI of one representative long-term survivor mouse from Fig. 5d 45 days post tumor rechallenge. White arrows indicate the needle track of the rechallenge injection site

HSV-P10 modulates virally induced PD-L1 expression

PTEN loss has been shown to correlate with PD-L1 upregulation^35,41–43, and resistance to T-cell mediated therapies³⁵. Thus we hypothesized that overexpression of PTENα in tumor cells had the potential to enhance T-cell response to therapy via downregulation of cell-surface PD-L1. To test this, we infected a panel of tumor cell lines with HSVQ or HSV-P10 and measured cell surface PD-L1 expression using flow cytometry (Fig. 7a, b). HSVQ virus infection of tumor cells revealed an induction of cell-surface PD-L1 expression on three of four breast cancer cell lines tested. HSV-P10 treatment showed a reduction of PD-L1 expression relative to HSVQ in all four cell lines tested (Fig. 7a, b). Consistent with the observation of increased PD-L1 observed in melanoma patients receiving oncolytic HSV virotherapy¹⁰, HSVQ virus infection of tumor cells revealed an induction of cell-surface PD-L1 expression on 3 of 4 breast cancer cell lines tested. HSV-P10 treatment showed a reduction of PD-L1 expression relative to HSVQ in all four cell lines tested (Fig. 7a, b). Analysis of PD-L1 expression in GFP negative uninfected cells revealed a significant induction of PD-L1 expression in cells adjacent to control HSVQ-infected cells. This increased expression of PD-L1 in adjacent uninfected cells was significantly mitigated in cultures treated with HSV-P10 (Fig. 7c). Since PTENα is thought to retain both lipid phosphatase and mitochondria modulating functions we evaluated the contribution of PTENα’s lipid phosphatase function in modulating tumor cell expressed PDL-1. Briefly, DB7 tumor cells were infected with HSVQ or HSV-P10 with or without LY294002, a PI3K antagonist that can reduce pAKT levels, and were evaluated for cell surface PDL-1 expression by fluorescent associated cell cytometry (gating strategy shown in Supplementary Figure ⁴). Consistent with Fig. 7a, we observed increased PDL-1 expression upon HSVQ infection (Fig. 7d left panel). This induction of cell surface PDL-1 was significantly rescued when cells were treated with HSVQ in combination with the LY294002. PI3K inhibition did not further reduce PDL-1 expression in tumor cells infected with HSV-P10 (Fig. 7d right panel). These results demonstrate that HSV-P10 mediated downregulation of PDL-1 expression is mediated by its ability to modulate PI3K effect.

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Fig. 7

HSV-P10 reduces cell-surface PD-L1 expression. a Histograms of PD-L1 expression as measured by flow cytometry 16hpi in a panel of human and mouse cancer cell lines. Gates were placed on live cells, and then for GFP positive cells. Relative fluoresence intensity was calculated based upon PD-L1 stain. Red histogram: isotype control, blue histogram: no virus, orange histogram: HSVQ, green histogram: HSV-P10. b Quantification of relative median fluorescence intensity (RFI) of the indicated cells treated with no virus, HSVQ or HSV-P10 and stained with PDL-1 antibody where statistical significance was assessed by one-way ANOVA (n=3/group, *p<0.05, ***p<0.001, ****p<0.0001). c Changes in PD-L1 expression (±s.d.) on uninfected (GFP negative) DB7 cells following low-MOI infection with either HSVQ or HSV-P10 where statistical significance was assessed by one-way ANOVA (n=3, *p<0.05, ****p<0.0001). Gates were placed on live cells, and then for GFP negative cells. Relative fluoresence intensity was calculated based upon PD-L1 stain. d Changes in PD-L1 expression measured by flow cytometry 12hpi in infected DB7 cells treated with HSVQ±PI3Ki or HSV-P10 (LY294002). Gating schematics are outlined in supplementary figure ⁵

HSV-P10 recruits innate and adaptive immune effector cells

Because herpesvirus infection, as well as reduced PD-L1, are known to be strong drivers of immunogenic activation^{10,17,44–49}, we hypothesized that HSV-P10 infection would have a strong effect on immune cell recruitment and activation in vivo. To evaluate if HSV-P10 altered the immune responses after oncolytic HSV infection, we compared immunohistochemistry (IHC) samples of tumor-bearing mice brains scarified 3 days after virus treatment, which revealed increased immune cell recruitment around virus-treated tumors. H&E staining revealed a gross reduction in tumor burden following HSV-P10 treatment (Fig. 8a). IHC staining for tumor cells expressing cytokeratin revealed the extent of tumor destruction following HSV-P10 treatment (Fig. 8b). Note the absence of cytokeratin 8 (epithelial cell marker) positive tumors in HSV-P10 treated mice 3 days after treatment, highlighting the increased tumor clearance. Both HSVQ and HSV-P10 treated tumors recruited F4/80+macrophages or microglia. F480 stained cells at these inflamed rims appeared activated with a bushy phenotype which differed from the ramified cells observed at the border between the brain and tumor in the saline treated control group. Both HSVQ and HSV-P10 treatments induced NK cell recruitment (black arrow heads highlight areas of NK cell influx), and staining was observed closely surrounding infected tumors. However, the total number of infiltrating NK cells was much lower in comparison to other immune cell types. CD3+and CD8+T-cells were observed preferentially infiltrating the infected tumor mass or tumor remnants, as well as diffusely throughout glial scarring left in the wake of rapid oncolytic tumor lysis. Little to no T-cell staining was observed at distal sites within the brain, indicating that the T-cell response was localized to areas of tumor cell implantation or virus injection. T-cell influx was more pronounced and localized around the microscopic tumor in HSV-P10 treated mice brains, while HSVQ treated tumors had inconsistent areas of high (depicted) T-cell infiltration. We observed a pattern of PD-L1 upregulation in HSVQ treated tumor cells 24hpi in vitro; however, PD-L1 was not detected in high levels 72hpi in any of the treatment groups in vivo due to the rapid clearance of virus in this model (Fig. 8b). Both HSVQ and HSV-P10 viruses were observed at detectable levels by both PCR and viral titrations in the brain 24hpi indicating that PTENα had no antagonist effects on viral replication in vivo (Supplementary Figure ⁵Figure).

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Fig. 8

HSV-P10 induces enhanced immune cell influx towards infected tumors. Tumor-bearing FVB/N mice were treated with 1e5 pfu of HSVQ, HSV-P10, or saline control 8 days post tumor implantation. a Stitched 4X bright field images of H&E stained sagittal sections of mouse brains 3 days post virus injection. b Representative 20X bright field images of H&E, Keratin 8, F4/80, NKp46, CD3, CD8α, PD-L1, and HSV1 (GFP) from sagittal sections of mouse brains 3 days post virus injection. Scale=0.1mm. c Ratio of macrophages (CD11b+F4/80+CD45^bright) to microglia (CD11b+F4/80+CD45^dim). d Percentage of cells in c positive for MHC-II. e Dendritic cell (CD11c+CD80+MHC-II+) infiltration. f NK cell (CD49b+NKp46+) infiltration. g CD8+T-cell (CD3+CD4+) infiltration. h CD4+T-cell (CD3+CD8+) infiltration. Data shown are averages±s.d (n=3/group) Statistical significance was assessed by one-way ANAOVA (n=3, *p<0.05, ***p<0.001, ****p<0.0001). Gating strategies for (c–h) are decribed in supplementary figure ⁶

To quantitatively evaluate immune cell recruitment and activation in virally treated animals, we evaluated infiltration of macrophages, NK cells, dendritic cells, and CD4 and CD8 positive T-cells within the tumor-bearing brain hemisphere 7 days post HSVQ or HSV-P10 treatment via flow cytometry (gating strategies depicted in Supplementary Figure ⁶Figure). Flow cytometric analysis revealed an influx of peripheral immune cells into the brains of virally infected animals compared to saline treated controls (Fig. 8c–h). Treatment with HSV-P10 induced significantly greater macrophage infiltration (CD11b+, F4/80+, CD45^bright) (Fig. 8c) and MHC-II presentation on both resident microglia (CD11b+, F4/80+, and CD45^dim) (Fig. 8d, blue) and infiltrating macrophages (Fig. 8d, red). HSV-P10 also induced significantly greater dendritic cell (CD11c+, CD80+, MHC-II+) (Fig. 8e), NK-cell (CD49b+, NKp46+) (Fig. 8f), and CD8+T-cell (CD3+, CD8+) (Fig. 8g) infiltration over saline control and HSVQ-infected animals. At 7 days post virus infection, ~2.5% of the immune cells in HSV-P10 treated brains were NK cells, and 10% were CD8+T-cells. Conversely, HSVQ treated animals recruited approximately 1% NK cells and 4% CD8 T-cells, neither of which differed significantly from saline treated control mice. HSVQ infection induced a significant influx in macrophage infiltration (Fig. 8c), and both HSV-P10 and HSVQ treatment resulted in a significant reduction in CD4+T-cell (CD3+, CD4+) infiltration (Fig. 8h) into infected brain hemispheres. B-cells (CD19+) and myeloid derived suppressor cells (CD11b+Gr1+) were not detected in any of the treatment groups (data not shown). We therefore assessed the B-cell response via antibody production. To test changes in an antibody-mediated response against virus, tumor, or infected tumor, serum from mice harvested 7 days post virus infection (Supplementary Fig. ^7a) was tested for antibody reactivity against purified virus lysate (Supplementary Fig. ^7b), DB7 tumor cell lysate (Supplementary Fig. ^7c), or HSVQ-infected tumor cell lysate (Supplementary Fig. ^7d). While virus infection increased reactivity against tumor, virus, and infected tumor cells, both the viruses induced equivalent anti-virus and antitumor antibody responses.

Virus purification

A total of 10–25 150mm dishes were seeded overnight with Vero cells at ~80% confluence in 2% FBS-containing DMEM at 37°C. Seeded cells were infected with virus (either HSVQ or HSV-P10) at low multiplicity of infection (MOI) and incubated at 34°C until ready for virus harvest. Cells were pelleted at 500× g in a tabletop centrifuge, and cell-containing media was subjected to 3 freeze-thaw cycles (liquid nitrogen, 37°C water bath) to release virus from cells. Cell debris was pelleted at 4000 RPM and supernatants were combined and transferred to 250mL centrifuge containers where the virus was pelleted at 13,000 RPM using a floor-standing centrifuge. Virus pellets were resuspended in sterile saline, filtered through a 5μm Millex PVDF filter, and overlaid on 30% sucrose in sterile saline in Oak Ridge tubes. Virus was re-pelleted at 11,000 RPM using a floor-standing centrifuge, and pellets were resuspended in sterile saline supplemented with 10% glycerol, aliquoted, and stored at −80°C.

Virus titration

Virus concentration was measured using a plaque forming assay. Purified virus was serially diluted and added to a 96-well plate. Virus-containing wells were overlaid with 10,000 Vero cells per well and incubated at 37°C overnight. ≤16h after Vero cell overlay, pooled human IgG (Grifols) was added at a final concentration of 1μg/mL, and plates were transferred to 34°C for 48–72h to develop plaques. Plaques were counted using fluorescence microscopy, and virus concentration was calculated as plaque forming units (PFU)/mL.

Cell lines

DB7, Met-1, and MVT-1 murine breast cancer and MDA-MB-231, SK-BR-3, MCF-7, and MDA-MB-468 human breast cancer cells were kindly donated by the lab of Dr. Mike Ostrowski at Ohio State University. U251 cells were obtained from Dr. Erwin G. Van Meir (Emory University, Atlanta, GA), and U251-T3 cells were created in our laboratory (May 2009) as a tumorigenic clone of U251 cells by serially passaging these cells three times in mice. LN229 and U87ΔEGFR cells were obtained from Erwin G. Van Meir (Emory University, Atlanta, GA). U87 cells are listed on the ATCC web site as as “likely glioblastoma cells” of male origin. Human Neural stem cells were obtained from Invitrogen (Carlsbad CA) and were differentiated into neurons per manufacturer protocols. HUVEC were obtained from ScienCell Research Laboratories Carlsbad, CA. U87ΔEGFR, U251-T3, and LN229 human glioblastoma cell lines were verified by short tandem repeat (STR) profiling to match DSMZ-held stocks at all loci on 1/14/2015. Vero cells were purchased from ATCC in 2013. U87ΔEGFR, U251-T3, and LN229 reporter cell lines were generated using lentiviral transduction of either a reporter construct or control construct, followed by puromycin selection for transduced clones. All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin antibiotic. All cells are routinely STRS profiled, maintained at a passage below 50, and checked for mycoplasma. The UTHSC IRB has determined that these studies qualify for exempt status.

Animals

All animal experiments were in compliance relevant ethical regulations and were conducted with the approval of the Ohio State University and University of Texas Health Science Center IACUC. For intracranial surgery, mice were anesthetized, heads shaved (if necessary), and stabilized in a stereotactic device (Kopf). The surgical site was sterilized with betadine/isopropyl alcohol, and a midline incision was made in the scalp. A 1mm burr hole was drilled 2mm lateral and 1mm anterior to bregma, and needles (Hamilton 80300 for cells or Hamilton 80000 for virus) were lowered 3mm into the brain and injected in a 2µL volume over 5min using autoinjectors (KD Scientific). Needles were removed, burr holes were filled with bone wax, and scalps were sutured using nylon sutures.

For survival studies, 50,000 DB7 breast cancer or U87ΔEGFR cells were implanted into the brains of 6–8 week old immunocompetent FVB/N (Jackson Labs) or immunocompromised athymic NUDE mice (TVSR, Ohio State University), respectively. Seven days after tumor cell implantation, mice were treated intratumorally with sterile saline control, or 1e5 PFU of either HSVQ or HSV-P10.

For T-cell depletion, mice were injected intraperitoneally with 0.5mg of isotype control (Bio-X-Cell BE0090), anti-CD4 (Bio-X-Cell BE0119), or anti-CD8a (Bio-X-Cell BE0117) antibodies (Supplementary Data ¹) 2, 4, and 7 days following virus injection following previously published protocols⁶⁶. Mice were then monitored for overall survival.

For tumor rechallenge, long-term survivor FVB/N mice (>90 days after tumor implantation) were imaged by MRI for the presence or absence of brain tumor. Mice with no indication of tumor burden were inoculated with 100,000 DB7 tumor cells into the left (contralateral) brain hemisphere alongside age-matched control mice. Mice were then followed for overall survival, and a follow-up MRI was performed 45 days after tumor rechallenge.

For immunohistochemistry, immune cell flow cytometry, and in vivo virus titration, 6–8-week-old FVB/N mice were intracranially inoculated with 100,000 DB7 cells as described. Eight days after tumor cell implantation, tumors were injected with saline control, or 1e5 PFU of either HSVQ or HSV-P10 in a volume of 2µL. One, three, or seven days after virus injection, mice were euthanized and brains were extracted. For immunohistochemistry, whole brains were fixed with 4% paraformaldehyde and stored at 4°C for 48h. For immune cell flow cytometry and in vivo virus titration, tumor-bearing hemispheres were separated from non tumor-bearing hemispheres, and tumor-bearing hemispheres were immediately placed in PBS. Virus titrations were performed on homogenized, freeze/thawed tumor-bearing brain hemispheres using the supernatant. Mononuclear cells were isolated following previously described protocols⁶⁷ for flow cytometric analysis.

For splenocyte isolation, spleens were dissected from euthanized mice and immediately placed in 10% FBS-containing DMEM. Splenocytes were extracted from spleens using the plunger end of a 1mL syringe, and splenocytes were passed through a 70µm cell strainer. Strained cells were pelleted, red blood cells lysed in RBC lysis buffer, and re-pelleted to remove lysis buffer and cell debris.

Western blot

Cultured cells were infected with HSVQ, HSV-P10, or saline at the indicated MOI for the indicated time. Cell lysates were harvested in RIPA cell lysis buffer with protease and phosphatase inhibitors added prior to use. Lysed cells were sonicated and cell debris removed via centrifugation. Protein concentration was measured by BCA and normalized prior to loading in precast gels. PVDF membranes were exposed to antibodies following manufacturer’s protocol. Primary (from CST: PTEN (9559), Akt (4691), pAkt-S473 (9271), from abcam: HSV1 ICP4 (ab6514), GAPDH (ab9484)) and secondary antibodies (from abcam: anti-mouse HRP (ab6789), anti-rabbit HRP (ab6721) can be found in Supplementary Data ¹. All phospho-antibody incubations were performed overnight at 4°C, and all non-phospho-antibody incubations were performed at RT for 1–2h. Uncropped blots are depicted in Supplementary Figure ⁹.

Immunocytochemistry

DB7 or Met1 cells were seeded on glass-bottomed tissue culture dishes overnight. Seeded cells were infected at MOI=0.1 with either HSVQ or HSV-P10 for 12h. Infected cells were washed 3×, fixed/permeabilized, and stained for pAkt-S473 using an anti-pAKT primary (CST 4060) and AF-594 tagged secondary (Thermo Fisher A-11012) antibody.

Cell surface and intracellular flow

For immune cell profiling of tumor-bearing brain hemispheres, mice were euthanized and whole brains were grossly dissected. The tumor-bearing brain hemisphere was dissociated by filtration through a 40μm cell strainer, and immune cells were isolated in a percoll gradient. Immune cells were then stained for a variety of markers for differentiation (Supplementary Data ¹). Macrophages/microglia/myeloid derived suppressor cells were stained using antibodies against CD11b (Miltenyi 130-096-834), F4/80 (Miltneyi 130-102-422), CD45 (Miltenyi 130-102-430), Gr1 (Miltenyi 130-102-385), and MHC-II (BD 563413). B and NK cells were stained with CD19 (Miltenyi 130-102-494), CD49b (Miltenyi 130-102-337) and NKp46 (Miltenyi 130-102-347). T-cells were stained with CD3 (Miltenyi 130-102-793), CD4 (Miltenyi 130-102-541), and CD8 (Mitenyi 130-102-807), and dendritic cells were stained with CD11c (MItenyi 130-102-545), CD80 (BD 560523), CD86 (Miltenyi 130-102-558), and MHC-II BD 563413). Stained cells were analyzed using a flow cytometer (BD LSR-II).

For splenocyte sorting, isolated splenocytes were stained for CD3 (Miltenyi 130-102-793) and NKp46 (Miltenyi 130-102-347) (Supplementary Data ¹), and sorted using a flow cytometer (BD FacsAria II).

For intracellular flow cytometry, U87ΔEGFR cells were seeded overnight, and infected at MOI=0.2 with either HSVQ or HSV-P10 for 8h. A volume of 5μM BKM120 (PI3K inhibitor) was used 1h as a negative control. Treated cells were fixed/permeabilized and stained using an anti-pAKT S473 V450 antibody (BD 560858) (Supplementary Data ¹). Stained cells were analyzed using a flow cytometer (BD LSR-II).

FACS sequential gating/sorting strategies are shown for Fig. 5 d,e in supplementary figure ², for Fig. 7d in supplementary figure ⁵, and for Fig. 8c-h in supplementary figure ⁶. FACS gating strategies are described verbally in the figure legends of Fig. 7 a,b and of Fig. 9b.

Viral infection and replication assay

Virus titration was performed on Vero cells using a plaque forming assay. In vitro, tumor cells were seeded overnight and infected at a MOI of 0.005. Cells and media were harvested 72hpi. Cells were lysed either by sonication or freeze/thaw cycles, and cell debris was pelleted and removed. Serial dilutions of virus-containing media were overlaid with Vero cells and incubated at 37°C for≤16h. Pooled human immunoglobulin was added at 1μg/mL final concentration, and cells were transferred to 34°C for 48–72h to develop plaques. In vivo, FVB/N mice were intracranially inoculated with 100,000 DB7 cells as described. Eight days after tumor cell implantation, tumors were injected with a single dose of 1e5 PFU of either HSVQ or HSV-P10. Mice were euthanized, and tumor-bearing brain hemispheres were collected, homogenized via filtration through a 70µm cell strainer, and sonicated for viral release. Cell debris was removed via centrifugation and supernatant was serially diluted and assayed as described above.

To measure infection and virus replication over time, cells were seeded at a density of 5×10⁴ cells per well in 24-well plates overnight. Seeded cells were infected with 0.05 MOI of HSVQ or HSV-P10, and virus infection was quantified by GFP expression, which was monitored utilizing the Cytation 5 Cell Imaging Multi-Mode Reader (Biotek) where images were taken every 2h.

Cell proliferation assay

Cells were seeded in 96-well plates containing 5000 cells/well in 2% FBS-containing media. Seeded cells were infected at MOI=0.5 with either HSVQ or HSV-P10 in 2% FBS-containing media. Cells and virus were incubated for 24–96hpi at 37°C, and cell viability was measured using a 2-step MTT assay (Roche) following manufacturer’s protocol. Absorbance was measured using a spectrophotometer (BMG Labtech FLUOstar Optima), and cell viability was measured as the percentage of uninfected control.

ATP measurement

Tumor cells were seeded in 24-well plates containing 100,000 cells per well in 2% FBS-containing media. Cells were allowed to adhere to tissue culture plates overnight at 37C, and were infected at MOI=0.5 with HSVQ or HSV-P10 for 1h. Unbound virus was removed and media was replaced. 24hpi, conditioned media was harvested and ATP concentration was assessed using an ATP determination kit (Thermo Fisher). Cell lysates were collected in RIPA buffer with protease and phosphatase inhibitors, and protein concentration was determined by BCA assay (Pierce, Thermo Fisher). Equal amounts of protein were then analyzed for ATP concentration as described. For OV±AKTi measurements, cells were seeded in 24-well plates at a density of 200,000 cells/well in 2% FBS-containing media, and infected at MOI=0.1 with HSVQ or HSV-P10 for 1h. Unbound virus was removed and replaced with media±5µM LY294002 (a PI3K inhibitor that reduces AKT phosphorylation). Conditioned media was harvested 12hpi and ATP was measured via CellTiter-Glo 2.0 (Promega).

⁵¹Cr release assay

Splenocytes from naive or immune animals were harvested and sorted as described. CD3+cells were stimulated by CD3/CD28 beads (Gibco) and recombinant IL2, and co-cultured with ⁵¹Cr labeled uninfected DB7 cells. Maximum (SDS treatment) and minimum (no co-culture) ⁵¹Cr release controls were included for analysis, and cells were incubated at 37C overnight. Cells were then collected via centrifugation and supernatants were measured for ⁵¹Cr concentration using a scintillation counter.

ELISA

For antibody titrations against virus, infected DB7 cells, and uninfected DB7 cells, mouse serum was harvested from tumor-bearing FVB/N mice 7 days post virus treatment by submandibular bleed, followed by coagulation and separation of whole blood, and collection of serum. Opaque 96-well plates were coated with antigen in carbonate/bicarbonate buffer overnight at 4°C, and washed 4 times with PBST. Serum was diluted in 5% skim milk/PBST 1:100, incubated 2h at RT. Plates were washed 4 times with PBST, and incubated with anti-mouse HRP at 1:1000 in 5% skim milk/PBST for 1h at RT. Plates were washed 4 times with PBST, and substrate added for quantification using a spectrophotometer (BMG Labtech FLUOstar Optima).

Immunohistochemistry

Dissected tissues were fixed in 4% paraformaldehyde solution for 48h, dehydrated in 30% sucrose for 24h, and transferred to 70% ethanol. Tissues were processed, embedded in paraffin, cut in 5μm sections on positively charged slides, de-paraffinized, rehydrated, and stained with H&E.

For immunohistochemistry, all sections were stained using a Bond Rx autostainer. Briefly, slides were heated at 65°C for 15min and the automated system performed dewaxing, rehydration, antigen retrieval, blocking, primary antibody (Cytokeratin 8 (Developmental Studies Hybridoma Bank, University of Iowa), CD31 (Santa Cruz sc-1506R), PTEN (CST 9559), F4/80 (Invitrogen MF48000), NKp46 (Abcam ab214468), CD3 (Abcam ab16669), CD8a (CST 98941), PD-L1 (CST 64988)) incubation, post primary antibody incubation, detectionco, and counterstaining using Bond reagents. Samples were then removed from the machine, dehydrated with ethanols and xylenes, mounted and coverslipped. Antibodies were diluted in antibody diluent, as indicated in Supplementary Data ¹.

Magnetic resonance imaging

Micro MRI imaging was conducted using a 9.4T BioSpec 94/30 Imaging System. T1 and T2 weighted images were captured and analyzed using Paravision 5.1 software. Screen captures of coronal brain sections at the site of tumor injection (as seen by needle track) were included for publication.

The images in Fig. 10 were drawn by Beth Watson and Cynthia Reyna in UTHealth’s Multimedia Scriptorium, an on campus support group for faculty and staff. Research reported in this publication was supported by the National Cancer Institute, National Institute Of Neurological Disorders And Stroke of the National Institutes of Health under Award Numbers: P01 CA163205, R01 CA150153, R01 NS064607, and F32NS108528, and The Pelotonia Fellowship to LR. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.