Abstract

   Rationale: The resistance of pancreatic ductal adenocarcinoma (PDAC) to
   immunotherapies is caused by the immunosuppressive tumor
   microenvironment (TME) and dense extracellular matrix. Currently, the
   efficacy of an isolated strategy targeting stromal desmoplasia or
   immune cells has been met with limited success in the treatment of
   pancreatic cancer. Oncolytic virus (OV) therapy can remodel the TME and
   damage tumor cells either by directly killing them or by enhancing the
   anti-tumor immune response, which holds promise for the treatment of
   PDAC. This study aimed to investigate the therapeutic effect of
   OX40L-armed OV on PDAC and to elucidate the underlying mechanisms.

   Methods: Murine OX40L was inserted into herpes simplex virus-1 (HSV-1)
   to construct OV-mOX40L. Its expression and function were assessed using
   reporter cells, cytopathic effect, and immunogenic cell death assays.
   The efficacy of OV-mOX40L was then evaluated in a KPC syngeneic mouse
   model. Tumor-infiltrating immune and stromal cells were analyzed using
   flow cytometry and single-cell RNA sequencing to gain insight into the
   mechanisms of oncolytic virotherapy.

   Results: OV-mOX40L treatment delayed tumor growth in KPC tumor-bearing
   C57BL/6 mice. It also boosted the tumor-infiltrating CD4+ T cell
   response, mitigated cytotoxic T lymphocyte (CTL) exhaustion, and
   reduced the number of regulatory T cells. The treatment of OV-mOX40L
   reprogrammed macrophages and neutrophils to a more pro-inflammatory
   anti-tumor state. In addition, the number of myofibroblastic
   cancer-associated fibroblasts (CAF) was reduced after treatment. Based
   on single-cell sequencing analysis, OV-mOX40L, in combination with
   anti-IL6 and anti-PD-1, significantly extended the lifespan of PDAC
   mice.

   Conclusion: OV-mOX40L converted the immunosuppressive tumor immune
   microenvironment to a more activated state, remodeled the stromal
   matrix, and enhanced T cell response. OV-mOX40L significantly prolonged
   the survival of PDAC mice, either as a monotherapy or in combination
   with synergistic antibodies. Thus, this study provides a multimodal
   therapeutic strategy for pancreatic cancer treatment.

   Keywords: oncolytic virus, pancreatic cancer, OX40L, tumor immune
   microenvironment, T cell

Introduction

   Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid
   tumors, with 466,003 deaths worldwide in 2020 [71]^1^, [72]^2. Despite
   advances in conventional chemotherapy and radiation, they have not been
   shown to significantly improve life expectancy in patients with PDAC.
   Similarly, immunotherapy has yet to be proven effective [73]^3.
   Resistance to immunotherapies in PDAC is caused by immunosuppressive
   tumor microenvironment (TME) and dense extracellular matrix [74]^4.

   Recent discoveries in immunology and the TME have inspired the
   development of oncolytic virotherapy [75]^5^-[76]^7. Oncolytic viruses
   (OVs) have the ability to convert immunologically “cold” tumors into
   “hot” ones by activating both innate and adaptive immunity [77]^8^,
   [78]^9. Talimogene laherparepvec (T-VEC), the first FDA-approved OV
   that was a granulocyte-macrophage colony-stimulating factor
   (GM-CSF)-armed herpes simplex virus-1 (HSV-1), could effectively reduce
   the tumor burden in both injected and non-injected melanoma lesions
   [79]^10^-[80]^13. Previous research reported that oncolytic HSV-1
   reprogrammed immunosuppressive TME to a more proinflammatory state,
   especially decreased anti-inflammatory macrophages [81]^9. In addition,
   CD40L-expressing HSV-1 therapy promoted dendritic cell (DC) maturation
   and cytotoxic T cell activation, significantly prolonging the survival
   of mice with PDAC [82]^14. Intratumoral delivery of transgenes through
   OV enables gene expression in situ, which is crucial for some
   immune-stimulating proteins because the systemic administration of
   these immune modulators is associated with severe adverse effects or
   weak immune responses at the tumor location.

   OX40 belongs to the tumor necrosis factor receptor superfamily (TNFRSF)
   and is widely expressed on activated T cells and neutrophils. The
   interactions between OX40 and OX40L act as co-stimulatory signals for
   T-cell activation that enhance the proliferation, survival, activation,
   and differentiation of CD4+ and CD8+ T cells and block the suppressive
   function of regulatory T cells (Tregs) [83]^15^, [84]^16. Furthermore,
   OX40L and OX40 agonists promote the activation and survival of
   neutrophils [85]^17^, [86]^18. Nevertheless, OX40 agonist therapy in
   pancreatic cancer is hindered by inefficient T cell trafficking into
   tumor immunosuppressive TME. DNX-2440, an OX40 ligand expressing
   oncolytic adenovirus, triggered an anti-tumor immune response and led
   to prolonged survival in preclinical models of cancers such as gliomas
   [87]^19. In addition, virus-like particles (VLPs) harboring OX40 ligand
   showed potential for boosting T-cell activation and eliminating tumor
   cells [88]^20. Nevertheless, HSV-1 has its own advantages for its
   bigger capability to insert multiple transgenes and more potent
   oncolytic activity. Furthermore, two HSV-1-derived virotherapy have
   been approved for marketing, and the efficacy and safety of HSV-1-based
   oncolytic therapies have been clinically validated [89]^21^-[90]^23.

   Hence, we engineered an HSV-1-based murine OX40L-expressing oncolytic
   virus (OV-mOX40L). OV-mOX40L exhibited local and systemic tumor
   suppression and prolonged the survival of PDAC mice. Single-cell RNA
   sequencing (scRNA-seq) revealed that OV-mOX40L treatment activated
   tumor-infiltrating CD4 and CD8 T cells and decreased Tregs, along with
   the reprogramming of macrophages and neutrophils to a more
   pro-inflammatory anti-tumor state. Moreover, OV-mOX40L remodeled the
   stromal matrix to promote T-cell infiltration. This study aimed to
   investigate the immunological effects of OV-mOX40L on
   tumor-infiltrating immune cells, which could potentially be used as
   monotherapy or in combination with synergistic antibodies for PDAC
   treatment.

Materials and Methods

Cells

   Vero cells were originally obtained from ATCC (CCL-81, ATCC). KPC cell
   line was purchased from Shanghai Biomodel Organism Science & Technology
   Development Co., Ltd, (Catalog number: NM-YD04). KPC mice were obtained
   by crossing Trp53-R172H, Kras-LSL-G12D with Pdx1-Cre-Tg. The KPC mouse
   contains a dominant negative point mutation (Trp53-R172H) in the P53
   gene and a conditionally activating point mutation (Kras-LSL-G12D) in
   the KRAS gene with a lox-stop-lox (LSL) termination sequence upstream.
   Cre-mediated recombination excises the LSL termination sequences and
   enables expression of KRASG12D in pancreatic tissue [91]^24. KPC cells
   derived from the KPC mice and were capable of tumor formation after
   subcutaneous inoculation in C57BL/6 mice. These cells were cultured in
   DMEM supplemented with 10% fetal bovine serum (FBS) and 1%
   penicillin/streptomycin and incubated at 37 °C with 5% CO[2].

Construction of oncolytic viruses

   The trimerized membrane-bound OX40L was constructed by linking a mouse
   TRAF2 coiled-coil domain to a mouse OX40L extracellular domain. An
   interleukin-2 (IL-2) signal sequence and a PDGFR transmembrane domain
   were added at the 5' and 3' ends, respectively. The expression
   cassettes were inserted into the ICP34.5 location of the HSV-1 genome
   to construct OV-mOX40L. The viruses were propagated, titered, and
   purified as previously described [92]^9.

Immunogenic cell death analysis

   Approximately 2×10^5 cells were mixed with the oncolytic virus at a
   multiplicity of infection (MOI) of 1. After 48 hours, the supernatant
   was collected to detect the ATP concentration using the Enhanced ATP
   Assay Kit (S0027, Beyotime). The tumor cells were stained with
   anti-calreticulin (CRT) antibody (bs-5913R-AF647, Bioss), and subjected
   to flow cytometry analysis.

Reporter cells assay

   Jurkat-NF-κB-GFP-OX40 reporter cells were constructed to detect the
   function of OV-mOX40L. Jurkat cells were stably transfected with a GFP
   reporter gene under the control of NF-κB response element and OX40
   sequences. Approximately 1×10^6 HEK293FT cells were mixed with the
   oncolytic virus at an MOI of 1. Twenty-four hours after infection, the
   infected HEK293FT cells were mixed with 5×10^6 Jurkat-NF-κB-GFP-OX40
   reporter cells for 24 hours of coculture. The reporter Jurkat cells
   were harvested and subjected to flow cytometry analysis to detect the
   expression of GFP.

Ex vivo assay

   Approximately 1×10^5 KPC cells were mixed with an oncolytic virus at an
   MOI of 1 and incubated for 24 hours. Mouse spleen was separated from
   C57BL6/J mice and ground into splenocytes, then CD3+ T cells or Tregs
   were sorted by magnetic beads (130-094-973, 130-095-925, Miltenyi),
   respectively. The cell culture medium of KPC infected with the
   oncolytic virus was replaced with RPMI1640 complete medium
   (RPMI1640+10% FBS+2% penicillin/streptomycin) and added 5×10^5 isolated
   T cells or Tregs, cocultured for 24 hours. Then T cells were harvested
   and stained with CD4, CD8, IFN-γ, and granzyme B antibodies, and
   subjected to flow cytometry analysis. The Treg cells were harvested and
   subjected to RNA extraction and reverse transcription. Foxp3 expression
   was detected by quantitative real-time PCR. For T cell proliferation,
   5×10^5 isolated CD3+ T cells were labeled with carboxyfluorescein
   succinimidyl ester (CFSE) and cocultured with 1×10^5 OV-infected KPC
   cells in a 96-well plate precoated with anti-CD3 antibody for three
   days. The proliferation of T cells was determined by flow cytometry.

Mice model

   The C57BL/6J mice were obtained from the Vital River Laboratory Animal
   Technology (Beijing, PR China). All animal studies were performed in
   accordance with the Institute Research Ethics Committee of Nankai
   University (Protocol Registry Number: A-2018-0306).

   For the KPC syngeneic tumor mouse model, approximately 5×10^6 KPC cells
   were mixed with Matrigel (v:v=1:1) and subcutaneously implanted into
   the right flank of 6-week-old male mice. Once the tumor volumes reached
   100-150 mm^3 (day 0), the tumor-bearing mice were treated
   intratumorally with 2×10^6 PFU of purified oncolytic virus per mouse on
   days 0, 3, 6, 9, and 12. For the combination therapy, anti-IL6 (BE0046,
   Bioxcell) and anti-PD-1 (BE0146, Bioxcell) neutralizing antibodies were
   injected intraperitoneally on days 0, 3, 6, 9, and 12.

   Tumor length and width were measured twice per week using a digital
   caliper, and tumor volumes (V, mm^3) were calculated using the formula:
   0.5×(length×width^2). Mice were euthanized when the tumor volume
   reached 2,000 mm^3. The survival of tumor-bearing mice was monitored
   and analyzed using Kaplan-Meier curves.

Flow cytometry analysis

   The mice were sacrificed three days after the last treatment. Tumor
   tissues were collected and cut into pieces. The single cell suspension
   was prepared by using dissociation buffer (1 mg/mL collagenase IV, 1
   mg/ml hyaluronidase, and 20 U/mL DNase in phosphate buffer saline (PBS)
   plus 2% FBS). The red blood cells were removed by incubating with ACK
   lysis buffer (CS0001, Leagene). The single cell suspension was stained
   with Zombie NIR™ Fixable Viability Kit (423105, BioLegend) and immune
   cell markers ([93]Table S1). Flow cytometry analysis was performed on
   an LSR Fortessa flow cytometer (BD Biosciences), and data were
   processed using FlowJo v10 software (TreeStar Inc., USA).

Single cell sequencing

   The single cell suspension was prepared as described above and CD45
   positive cells were isolated using CD45 microBeads (130-110-618,
   Miltenyi). Three samples from the same group were incubated with
   different hashtag antibodies (TotalSeq-C0301, C0302, C0303, Biolegend),
   washed and mixed in equal numbers. The single-cell sequencing was
   performed using Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10x
   Genomics) following the manufacturer's protocol. Sequencing was
   performed on NovaSeq 6000 (Illumina).

Identification of cell types and cluster marker genes in Seurat

   Gene barcode matrices were imported into the Seurat (version 4.0.5)
   pipeline for quality control and downstream analysis [94]^25. The
   HTODemux function in Seurat, using its default parameters, was used to
   assign each individual cell back to its original sample based on the
   normalized cell hashtag counts. Low-quality cells (< 100 genes/cell, >
   5,000 genes/cell, > 20,000 UMIs/cell, > 8% mitochondria) were excluded.
   The data were then normalized, and highly variable genes were detected
   using the FindVariableFeatures function. Principal component analysis
   was performed to reduce the dimensionality of the scRNA-Seq dataset.
   The top 71 principal components, which explained 90% of the variance in
   the dataset, were used to perform the downstream analysis. The Harmony
   package (version 1.0) was used to perform the batch correction [95]^26.
   Cell clusters were identified using the FindClusters function in Seurat
   with a resolution of 0.7. They were then visualized using t-distributed
   stochastic neighbor embedding plots. Each cell cluster was manually
   categorized into a known biological cell type.

   The FindMarkers function in Seurat was used to identify
   cluster-specific marker genes or differentially expressed genes in an
   individual cluster. The average expression of the genes within each
   cluster was calculated, and heatmaps of the average expression of genes
   were generated using the R package ComplexHeatmap (version 2.8.0).

Pathway enrichment analysis

   Gene set enrichment analysis (GSEA) was performed using WebGestalt
   [96]^27 to identify the enriched pathways annotated by Gene Ontology,
   KEGG, and Reactome databases. Pathway size was limited to 500 genes per
   pathway, and genes were ranked by log 2-fold change calculated by the
   Findmarkers function in Seurat where 1,000 permutations were used to
   estimate the FDR for the GSEA analysis.

Cell communication network construction

   The R package CellChat (version 1.1.3) was used to predict the
   potential cell interactions between the annotated cell clusters after
   the Seurat analysis [97]^28. Two CellChat objects for the PBS and the
   OV-mOX40L treatment groups were generated by the createCellChat
   function using the Seurat object. Cell communication networks were
   constructed by the computeCommunProb, computeCommunProbPathway, and
   aggregate net functions. Interaction comparison analysis was performed
   by the compareInteractions and RankNet function to detect the
   differentially increased cell interactions in the OV-mOX40L treatment
   group. The netAnalysis_signalingRole_heatmap and netVisual_aggregate
   functions were applied to generate the heatmap and circle plots of the
   interactions among specific cell clusters in each treatment group.

Statistical analysis

   Quantitative data were presented as the mean ± standard deviation (SD)
   of at least three independent experiments. Data were analyzed using
   one-way analysis of variance (ANOVA) with Dunnett test. Animal survival
   was plotted using Kaplan-Meier curves and compared using the log-rank
   test. A p-value less than 0.05 was considered to be statistically
   significant (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Results

Generation and characterization of OX40L-armed oncolytic HSV-1

   Considering that TRAF2-derived coiled-coil domain could stabilize OX40L
   trimerization [98]^29, we constructed trimerized mOX40L consisting of
   the IL2 signal sequence, Traf2 coiled-coil domain, OX40L extracellular
   domain, and PDGFR transmembrane domain. We evaluated the agonistic
   potential of trimerized mOX40L and whole natural mOX40L using
   Jurkat-NF-κB-GFP-OX40 reporter cells, which express GFP upon activation
   of the OX40-OX40L signaling pathway. Trimerized mOX40L-transfected
   HEK293FT showed slightly elevated reporter cell activation compared to
   natural mOX40L-transfected HEK293FT cells ([99]Figure S1). Therefore,
   we utilized trimerized mOX40L for the oncolytic virus modification. To
   generate OV-mOX40L, the trimerized mOX40L was inserted into the
   backbone of ICP34.5 and ICP47 double-deleted oncolytic HSV-1 (Figure
   [100]1A). Vero cells were infected with OV-mOX40L and
   membrane-displayed OX40L was determined by flow cytometry (Figure
   [101]1B). The pancreatic cancer cell line KPC was infected with either
   OV-GFP or OV-mOX40L, and the modification of the OV did not attenuate
   their replication capability (Figure [102]1C) and tumoricidal activity
   (Figure [103]1D). Induction of immunogenic cell death (ICD) was
   measured by the release of ATP and surface CRT expression according to
   previous studies [104]^30^-[105]^33. Although the release of ATP
   increased after oncolytic virus infection, the difference between
   OV-mOX40L and OV-GFP infection was minor (Figure [106]1E). The surface
   expression of CRT also increased after infection with OV-mOX40L (Figure
   [107]1F). In addition, the function of OV-mOX40L was assessed using
   Jurkat-NF-κB-GFP-OX40 reporter cells. In co-culture with the
   Jurkat-based reporter cells, the OV-mOX40L pre-infected HEK293FT cells
   stimulated the reporter cells more than the OV-GFP pre-infected
   HEK293FT cells (Figure [108]1G). To further evaluate the function of
   OV-mOX40L on T cells, we conducted ex vivo assays by isolating mouse
   splenic T cells and co-culturing them with OV-infected KPC cells. The
   CFSE assay showed that OV-mOX40L-infected KPC cells promoted the
   proliferation of T cells (Figure [109]1H). Furthermore, in comparison
   to OV-GFP-infected KPC cells, OV-mOX40L-infected KPC cells
   significantly increased the expression levels of IFN-γ and granzyme B
   (GZMB) in CD4+ T cells and CD8+ T cells, respectively (Figure [110]1I).
   These results indicated that OV-mOX40L treatment activated both CD4+
   and CD8+ T cells. Additionally, coculturing Tregs with
   OV-mOX40L-infected KPC cells slightly downregulated the expression of
   Foxp3 on Tregs ([111]Figure S2).

Figure 1.

   [112]Figure 1
   [113]Open in a new tab

   Generation and characterization of OX40L-armed oncolytic HSV-1. A) The
   schematic representation of HSV-1 based oncolytic virus encoding murine
   OX40L or GFP. The trimerized mOX40L was constructed by IL2 signal
   sequence-Traf2 coiled-coil domain-mOX40L extracellular domain-PDGFR
   transmembrane domain. B) GFP or membrane-displayed OX40L expression
   level on KPC cells after infection with OV-GFP or OV-mOX40L. KPC cells
   were infected with OV-GFP or OV-mOX40L at an MOI of 1. Cells were
   stained with OX40L antibody after 24 hours and detected by flow
   cytometry. C) Virus replication in the KPC cells. KPC cells were
   infected with OV-GFP or OV-mOX40L at an MOI of 0.1, and viral titers
   were determined using plaque assays at different times post infection.
   D) Cytopathic effect (CPE) of OV-mOX40L against KPC cells. KPC cells
   were infected with OV-GFP or OV-mOX40L at an MOI of 1. Cells were
   observed under the microscope after 24 hours. E-F) ICD of tumor cells
   was induced by OV-mOX40L. KPC cells were infected with OV-GFP or
   OV-mOX40L for 48 hours. The released ATP was detected by the ATP
   detection kit (E) and the cell surface exposure of calreticulin was
   analyzed by flow cytometry (F). The data for ATP concentration were
   representative of three independent experiments. G) Effect of OV-mOX40L
   on Jurkat-NF-κB-GFP-OX40 reporter cells. HEK293FT cells were infected
   with OV-GFP or OV-mOX40L and cocultured with Jurkat-NF-κB-GFP-OX40
   reporter cells, which express GFP upon activation of the OX40-OX40L
   signaling pathway. The GFP expression of the reporter cells was
   analyzed by flow cytometry. H) Effect of OV-mOX40L on T cell
   proliferation. CFSE-labeled T cells were cocultured with OV-infected
   KPC cells in 96-well plate precoated with anti-CD3 antibody for three
   days. The proliferation of T cells, which exhibited decreased CFSE
   fluorescence intensity, was detected by flow cytometry. I) Effect of
   OV-mOX40L on T cell activation. T cells were cocultured with
   OV-infected KPC cells in 48-well plate precoated with anti-CD3 antibody
   for 24 hours. The activation markers IFN-γ and granzyme B of T cells
   were detected by flow cytometry.

OV-mOX40L inhibited tumor growth in a syngeneic pancreatic cancer mouse model

   A syngeneic mouse model was used to assess the inhibitory effects of
   OV-mOX40L on pancreatic cancer growth. KPC cells were inoculated into
   C57BL6/J mice, which demonstrated intense desmoplasia and an
   immunosuppressive environment that mimicked the TME of PDAC patients.
   Tumor-bearing mice were treated with OV-GFP or OV-mOX40L (Figure
   [114]2A). Intratumoral administration of OV-mOX40L delayed the tumor
   growth rate and prolonged the survival of mice, with a median survival
   time of 60 days in the OV-mOX40L group and 53.5 days in the OV-GFP
   group, respectively (Figure [115]2B-D).

Figure 2.

   [116]Figure 2
   [117]Open in a new tab

   Anti-tumor effect of OV-mOX40L in syngeneic KPC pancreatic cancer
   model. A) Schematic of the regimen of oncolytic virus treatment. KPC
   were subcutaneously implanted into the flank of C57BL/6J mice. When the
   tumor size reached about 150 mm^3 (set as day 0; n=8 per group), the
   tumor-bearing mice were intratumorally injected with 2×10^6 PFU of
   OV-GFP or OV-mOX40L per mouse on day 0, 3, 6, 9, and 12. B) The tumor
   growth curves were shown until they exceeded 1,000 mm^3. The tumor
   volumes were measured twice every week. C) The Kaplan-Meier survival
   analysis is shown. Log-rank test was performed for statistical
   comparison of survival curves. D) The individual tumor growth curve of
   PBS, OV-GFP and OV-mOX40L groups. E and F) Anti-tumor activity of
   OV-mOX40L in bilateral KPC-bearing mouse model. KPC cells were
   implanted subcutaneously into left and right flanks of mice,
   respectively. PBS, OV-GFP or OV-mOX40L was intratumorally injected into
   right side tumors, and the growth of the injected primary tumor (E) and
   the non-injected distant tumor (F) was shown.

   Furthermore, we examined whether OV-mOX40L inhibited tumor growth at
   local and abscopal sites using a bilateral tumor-bearing mouse model.
   KPC tumor cells were implanted in both flanks of mice, and tumors on
   the right side were treated with OV-mOX40L, OV-GFP, or PBS and
   monitored for 35 days. In this experiment, both primary and distant
   tumor growth were reduced in the OV-mOX40L group compared to the OV-GFP
   group, indicating that virotherapy induced a systemic antitumor immune
   response (Figure [118]2E and F).

Local OV-mOX40L treatment reinvigorated intratumoral immune cells

   To explore the mechanism underlying OV-mOX40L treatment,
   tumor-infiltrating immune cells and cancer-associated fibroblasts
   (CAFs) were analyzed three days after the last treatment using flow
   cytometry.

   Treatment with OV-mOX40L reduced Treg counts and increased the
   proportion of proliferated CD4 T cells. Furthermore, the proportion of
   CD8+ T cells among CD45+ immune cells and the expression of IFN-γ and
   granzyme B (GZMB) in CD8+ T cells were moderately upregulated, and the
   number of exhausted CD8+ T cells with PD-1 and LAG-3 expression
   decreased (Figure [119]3A and [120]Figure S3A). This indicated the
   activation of CD8+ T cells and mitigation of CD8+ T cell exhaustion
   upon OV-mOX40L treatment.

Figure 3.

   [121]Figure 3
   [122]Open in a new tab

   Analysis of tumor-infiltrating immune cells and fibroblasts after the
   treatment with oncolytic viruses. Mice were subcutaneously implanted
   with KPC cells and treated with oncolytic viruses or PBS. Tumor tissues
   were collected 3 days after the last treatment. A-C) The single-cell
   suspension was prepared from tumor, stained with the fluorescent
   antibodies and subjected to flow cytometry analysis for the profiling
   of T cells (A), macrophages (B) and fibroblasts (C). Statistical
   significance was determined by ANOVA tests and multiple comparisons.

   Upon OV-mOX40L treatment, the number of tumor-associated macrophages
   (TAM), marked as CD11b+ F4/80+, did not change (Figure [123]3B and
   [124]Figure S3B). The pro-inflammatory M1-like macrophages (inducible
   nitric oxide synthase, iNOS+) increased, whereas the anti-inflammatory
   M2-like macrophages (CD206+) decreased. The results revealed a shift in
   the potential of macrophages from anti-inflammatory to
   pro-inflammatory.

   Flow cytometry showed that the number of fibroblast active protein
   (FAP)-expressing CAFs was significantly decreased by OV-mOX40L
   treatment. Anti-Ly6C and anti-MHCII antibodies were used to segregate
   FAP-positive CAF into three populations, including Ly6C-positive
   inflammatory CAFs (iCAF), MHCII-positive antigen-presenting CAFs
   (apCAFs) and MHCII/Ly6C-double negative myofibroblastic CAFs (myCAFs).
   The proportion of myCAFs and apCAFs over total CAF slightly decreased,
   whereas that of iCAF significantly increased (Figure [125]3C and
   [126]Figure S3C).

OV-mOX40L treatment activated conventional CD4+ T cells and CD8+ cytotoxic T
cells and reduced Treg proportion

   To further understand the immune cell populations associated with the
   anti-tumor response following OV-mOX40L treatment, tumor-infiltrating
   immune cells were subjected to scRNA-seq analysis. Twenty-one clusters
   were identified (Figure [127]4A) and characterized using a series of
   marker genes ([128]Figure S4A).

Figure 4.

   [129]Figure 4
   [130]Open in a new tab

   Single-cell RNA sequencing analysis of the tumor-infiltrating immune
   cells after OV-mOX40L treatment. A) All tumor-infiltrating immune cells
   from PBS and OV-mOX40L treated mice were clustered and showed as tSNE
   plots. Tumor-infiltrating immune cells were isolated from 3 mice per
   group and labeled with hashtag antibodies, then mixed in equal numbers
   for single-cell RNA sequencing. B) The proportions of intratumoral T
   and NK cells in the PBS and OV-mOX40L groups. C) GSEA pathway
   enrichment of OV-mOX40L versus PBS in individual CD8+ T cell, CD4+ T
   cell, NK and NKT cell clusters. D) The proportions of myeloid cells in
   the PBS and OV-mOX40L groups. E) GSEA pathway enrichment of OV-mOX40L
   versus PBS in individual neutrophils, macrophages and cDC clusters. F)
   Expression of pro-inflammatory marker gene (Nos2) and immunosuppressive
   marker genes (Spp1 and Mrc1) in the tSNE plot.

   Total T and NK cells included clusters of CD8+, CD4+, Treg, NK, NKT,
   and γδ T cells (Figure [131]4B). CD4+ T cells were defined as cd3d and
   cd4 positive cells. Treg cluster displayed a high expression of Foxp3,
   and proliferating CD4+ T displayed a high expression of Mki67.
   OV-mOX40L treatment significantly increased the proportion of
   conventional CD4+ T cells and reduced the Treg cell proportion (Figure
   [132]4B). CD8+ T clusters were defined as Cd3d, Cd8a double positive.
   Two CD8+ T clusters exhibited heterogeneous gene profiles: exhausted
   CD8+ T cluster (Pdcd1 and Lag3) and proliferating CD8+ T cells (Mki67).
   Following OV-mOX40L treatment, the proportion of activated CD8+
   cytotoxic T lymphocytes (CTL) slightly increased, whereas the
   proportion of exhausted CD8+ T cells dramatically decreased (Figure
   [133]4B). Pathway analysis revealed the upregulation of signaling
   pathways associated with cytotoxicity and T cell activation in both the
   cytotoxic T cluster and the exhausted CD8+ T cluster upon OV-mOX40L
   treatment (Figure [134]4C and [135]Figure S4B). The NK cluster was
   defined based on the expression of Nkg7 and Klrk1, while the NKT
   cluster was defined based on the expression of Ly6c2, Cd3d, Nkg7, and
   Klrk1 ([136]Figure S4A) [137]^34. Although the relative percentages of
   NK and NKT cells were moderately reduced by OV-mOX40L treatment (Figure
   [138]4B), pathway analysis revealed that both clusters upregulated the
   NK cell-mediated cytotoxicity pathway upon OV-mOX40L treatment (Figure
   [139]4C). Overall, these results indicated that CD4+ T, CD8+ T, and NK
   cells were activated by OV-mOX40L treatment.

OV-mOX40L treatment switched the tumor immune microenvironment to a more
pro-inflammatory state

   Myeloid cells, including DC, monocytes, neutrophils, and macrophages,
   were identified (Figure [140]4D). Neutrophils were defined based on the
   expression of S100a8 and S100a9 ([141]Figure S4A). Neu-Nos2 and
   Neu-Isg15 were defined as N1-like neutrophils with anti-tumor activity
   owing to their high expression of Nos2, Isg15, or Icam1
   [142]^35^-[143]^37, whereas Neu-Cxcr2 and Neu-Mmp9 were defined as
   N2-like neutrophils with pro-tumor activity and characterized by the
   expression of Cxcr2 or Mmp9 [144]^38^, [145]^39. OV-mOX40L treatment
   increased the proportion of N1-like neutrophils and decreased that of
   N2-like Neu-Mmp9 (Figure [146]4D). Treatment with OV-mOX40L induced
   antigen processing and presentation, cell killing, phagocytosis, and
   response to virus signal pathway, it also downregulated and focal
   adhesion pathway in neutrophils (Figure [147]4E and [148]Figure S4C).
   Moreover, neutrophils upregulated the expression of Nos2, which could
   contribute to their tumor-suppressive activity (Figure [149]4F).

   Macrophages were defined by the high RNA expression of Csf1r and Itgam,
   while monocytes were defined by Ly6c2 and Itgam's high expression and
   low expression of Itgax. The following three macrophage clusters
   exhibited heterogeneous gene profiles ([150]Figure S4A). Nos2-Mac
   displayed high expression levels of Itgam and Nos2. Mrc1-Mac displayed
   high expression of Adgre1 and Mrc1, and the gene signature of
   proliferation-Mac was similar to that of Mrc1-Mac, except for the high
   expression of Mki67, indicating that both clusters were
   immunosuppressive [151]^40^, [152]^41. Pathway analysis results
   revealed that macrophages were more pro-inflammatory after OV-mOX40L
   treatment than after vehicle control administration (Figure [153]4E and
   [154]Figure S4C). Moreover, OV-mOX40L treatment increased IFN-γ
   expression and induced macrophages to express higher levels of
   pro-inflammatory marker genes, such as Nos2 and reduced the expression
   of immunosuppressive marker genes, such as Spp1 and Mrc1 ([155]Figure
   S4D and Figure [156]4F).

   Two clusters of CAFs were identified based on the expression of Mfap2
   ([157]Figure S4A). The myCAF cells were characterized by high
   expression of collagens and contractile myofibroblast factors, such as
   Acta2, Tagln, and Col7a1 [158]^42. The iCAF cluster was characterized
   by the expression of chemokines such as Cxcl12. Treatment with
   OV-mOX40L increased iCAF levels and decreased myCAF levels ([159]Figure
   S4E).

Analysis of cell-cell communications in pancreatic cancer

   Unbiased cell-cell interaction inference analysis was performed across
   all immune cell types using CellChat to dissect global alterations
   [160]^28^, [161]^43. We analyzed the signaling pathways with the
   highest combined interaction strength ratios in the PBS and OV-mOX40L
   groups (Figure [162]5A). The signaling communication pathways of Cd226:
   Nectin2/Pvr, Tnf: Tnfrsf1a/Tnfrsf1b, TGFb: TGFbR/ACVR1, VEGF:
   Flt1/Flt4/Kdr, PDCD1: PD-L1 and IL6: IL6R/gp130, which were related to
   the poor prognosis, were upregulated with OV-mOX40L treatment,
   suggesting that these pathways could be potential targets for
   combination therapy with OV-mOX40L. The overall signal pattern of the
   clusters was further analyzed. After treatment with OV-mOX40L,
   interactions of the PDCD1: PD-L1 signaling pathway were observed in a
   variety of cell types, including T cells, macrophages, and neutrophils,
   whereas the IL6:IL6R/gp130 pathway was primarily observed in CAFs and
   macrophages (Figure [163]5B). Then, we focused on the PDCD1: PD-L1 and
   IL6:IL6R/gp130 pathways.

Figure 5.

   [164]Figure 5
   [165]Open in a new tab

   Identification of major signaling changes after the OV-mOX40L therapy.
   A) Normalized interactions were summed as the overall information flow
   and significantly different signaling pathways were identified between
   the PBS and OV-mOX40L groups. B) The overall signaling patterns among
   our defined cell clusters in the PBS group and the OV-mOX40L group. C
   and D) The inferred IL6:IL6R/gp130 (C) and PDCD1: PD-L1 (D) signaling
   network among the cell populations represented by the nodes. The edge
   width represents the interaction strength of the specific pathway.

   Intercellular IL6 signaling interactions among macrophages, DCs, Tregs,
   and iCAFs were present in the OV-mOX40L group, but only macrophage-iCAF
   interactions were observed in the PBS group (Figure [166]5C). Moreover,
   PD-L1 pathway interactions among immune cells were enhanced by
   OV-mOX40L treatment (Figure [167]5D). The upregulation of IL6
   [168]^44^-[169]^46 and PD-L1 [170]^47 pathways could negatively impact
   anti-tumor immunity, providing the rationale for combining OV-mOX40L
   with neutralizing antibodies against IL6 or PD-L1 pathways for the
   treatment of pancreatic cancer.

The combination therapy of OV-mOX40L with anti-IL6 and anti-PD-1 antibodies
prolonged the survival of PDAC-bearing mice

   We combined neutralizing antibodies against IL6 or PD-1 in OV-mOX40L to
   treat a mouse PDAC model (Figure [171]6A). Compared to OV-mOX40L or
   neutralizing antibody treatment alone, the combination therapy of
   OV-mOX40L with anti-IL6 or anti-PD-1 antibody significantly delayed
   tumor growth, and the most effective tumor inhibition was observed with
   the triple therapy (OV-mOX40L+anti-IL6+anti-PD-1) (Figure [172]6B). In
   a separate experiment, the survival of KPC-bearing mice was monitored.
   Median survival days in the OV-mOX40L and neutralizing antibody groups
   were extended from 39 to 61.5 and 45 days, respectively, compared to
   the basic control (PBS) group, while median survival was not achieved
   in the triple therapy group as 62.5% of the mice had been cured (Figure
   [173]6C and D). This finding corroborated that neutralizing antibodies
   against IL6 and PD-1 could enhance the effect of OV-mOX40L therapy.

Figure 6.

   [174]Figure 6
   [175]Open in a new tab

   The combination therapy of OV-mOX40L with anti-IL6 and anti-PD-1
   antibodies prolonged the survival of PDAC-bearing mice. A) Schematic of
   the regimen of oncolytic virus treatment. KPC were subcutaneously
   implanted into the flank of C57BL/6J mice. When the tumor size reached
   about 70 mm^3 (set as day 0; n=7 per group), the tumor-bearing mice
   were randomly divided into 8 groups and intratumorally injected with
   2×10^6 PFU OV-mOX40L or 100 μL PBS per mouse on day 0, 3, 6, 9, and 12.
   100 μg of each antibody present in each group was intraperitoneally
   injected per mouse on days 0, 3, 6, 9, and 12. B) The tumor volumes
   were measured every third day and presented by the tumor growth curve
   (n=7). C) Kaplan-Meier survival analysis of KPC-bearing mice is shown.
   KPC were subcutaneously implanted into the flank of C57BL/6J mice. When
   the tumor size reached about 100 mm^3 (set as day 0; n=8 per group),
   the tumor-bearing mice were randomly divided into 4 groups and
   intratumorally injected with 2×10^6 PFU OV-mOX40L or 100 μL PBS per
   mouse on days 0, 3, 6, 9, and 12. 100 μg of each antibody present in
   each group was intraperitoneally injected per mouse on day 0, 3, 6, 9,
   and 12. Log-rank test was performed for statistical comparison of
   survival curves. D) The tumor volumes were measured every 3 days and
   presented by the tumor growth curve (n=8).

Discussion

   In this study, a novel oncolytic HSV-1 expressing mOX40L was developed
   for the treatment of pancreatic cancer. This approach was designed to
   combine the direct oncolytic effect of OV with the immune-stimulating
   activity mediated by both OV and OX40L. Correspondingly, OV-mOX40L
   could effectively delay tumor growth and extend survival time in a
   syngeneic KPC mouse model compared to the parental oncolytic virus.

   Oncolytic viruses expressing various kinds of cytokines and immune
   checkpoints (ICs) have been evaluated in previous studies
   [176]^48^-[177]^50. For example, IL2-encoding oncolytic HSV
   (G47Δ-mIL12) significantly reduced the primary tumor volume and
   metastasis of triple-negative breast cancer (TNBC) [178]^51. Other
   studies have revealed that repeated administration of CD40L-armed HSV-1
   could improve the survival of PDAC-bearing mice and offer long-term
   protection from tumor relapse [179]^14. Therefore, OV can be used as a
   delivery platform and has broad clinical application perspectives.
   OX40, which mainly induces the activation and proliferation of T cells,
   has been considered a promising candidate target for cancer
   immunotherapy. It was reported that OX40 agonistic antibody could
   improve the survival of the glioblastoma mouse model and showed
   efficacy in clinical trials [180]^52^, [181]^53. Herein, we reported a
   modified HSV-1 characterized by the delivery of the immunostimulatory
   factor OX40L, which triggered OX40-OX40L signaling pathway-mediated
   responses. We engineered OV-mOX40L for PDAC treatment by combining the
   tumor-targeting effect of OV with the immune-potentiating effect of
   OX40L.

   According to the results of our single-cell transcriptomic analysis and
   flow cytometry of tumor-infiltrating immune cells, OV-mOX40L treatment
   decreased Foxp3+ Tregs, activated CD4+ and CD8+ T cells, and resulted
   in less exhausted CTLs. T cell activation mediated by OX40-OX40L
   interaction showed increased release of inflammatory cytokines such as
   IFN-γ, a crucial factor that polarizes macrophages toward the M1
   phenotype [182]^54. Under the synergistic or additive action of OV and
   OX40L, M2-like macrophages and protumor N2-like neutrophils showed
   reduced proportion and boosted anti-tumor immune responses, whereas
   pro-inflammatory N1-like neutrophils were remarkably increased after
   OV-mOX40L treatment. Thus, this study demonstrated that OV-mOX40L
   treatment was able to convert the immunosuppressive TME towards a more
   immunoreactive state and establish a more supportive immune niche to
   activate endogenous T cells.

   Stromal fibroblasts play a critical role in supporting tumor growth and
   drive immunosuppression. Thus, it is important to investigate the
   effect of OV on the stromal components of pancreatic cancer. Notably,
   this KPC model mimicked the intense extracellular matrix characteristic
   of pancreatic cancer. Additionally, flow cytometry results revealed
   that OV-mOX40L treatment significantly decreased CAFs.

   Currently, the efficacy of an isolated strategy targeting stromal
   desmoplasia or immune cells has been met with limited success for the
   treatment of pancreatic cancer. These results illustrate the complexity
   of the pancreatic cancer TME and suggest that combining complementary
   stroma-targeted and immune-cell-targeted modalities is a rational
   strategy. Our study confirmed the multifaceted role of the mOX40L-armed
   oncolytic virus in remodeling the desmoplasia and immunosuppressive
   tumor immune microenvironment of pancreatic cancer. Moreover, the
   results from our scRNA-seq analysis revealed that PDCD1: PD-L1 and
   IL6:IL6R/gp130 signaling interactions were enhanced between CAFs and
   macrophages as well as between neutrophils and T cells after OV-mOX40L
   treatment. OV-mOX40L, in combination with anti-IL6 and anti-PD-1,
   significantly extended the survival of KPC-bearing mice, indicating
   that combination therapy targeting CAFs and immune cells has
   considerable potential for clinical applications.

   We also observed that Cd226: Nectin2/Pvr, Tnf: Tnfrsf1a/Tnfrsf1b, TGFb:
   TGFbR/ACVR1 and VEGF: Flt1/Flt4/Kdr signaling pathways were upregulated
   upon OV-mOX40L treatment. Activation of these pathways could reduce the
   antitumor immune response; therefore, targeting them could potentially
   enhance the efficacy of OV-mOX40L. Actually, the power of scRNA-seq
   could guide the design of different combination therapy. For example,
   Gulhati et al. designed triple therapy with
   anti-4-1BB/anti-LAG3/CXCR1/2 inhibitor based on high-dimensional immune
   profiling and the combination therapy resulted in a durable and
   complete response [183]^55.

   In conclusion, the combination of the tumor-targeting OV and the
   immune-potentiating OX40L has shown promising results for the treatment
   of pancreatic cancer. OX40L-armed OV therapy can boost T-cell response
   and potentiate anti-tumor efficacy by remodeling the TME. The
   combination of OV-mOX40L and anti-IL6/anti-PD-1 antibodies remarkably
   prolonged the survival of PDAC-bearing mice. These findings provided
   important evidence for the efficacy and mechanisms of OV-mOX40L
   treatment and may therefore contribute to monotherapy or combination
   therapy of OV-mOX40L for PDAC treatment.

Supplementary Material

   Supplementary figures and table.
   [184]Click here for additional data file.^ (803.5KB, pdf)

Acknowledgments