2.1 Cell culture

SK-BR-3, MCF-7, and MDA-MB-231 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). EMT-6 cells were grown in RPMI-1640 containing 10% FBS. All cells were cultured in a humidified 5% CO₂ atmosphere at 37℃.

2.2 Cell viability assay

SK-BR-3, MDA-MB-231, MCF-7, and EMT-6 cells (10,000 cells/well) were seeded in 96-well plates and infected with VG161 at a multiplicity of infection (MOI) of 0, 0.01, 0.1, 1, or 10, or co-treated with 5 nM or 20 nM PTX, for the indicated times. Cells were then incubated with CCK-8 reagent (Beyotime, Shanghai, China) for 2 to 4 h. Absorbance values were measured at 450 nm using a plate reader (Bio-Rad).

2.3 VG161 and VG160 OV

Genes encoding human IL-12, IL-15, and IL-15 receptor alpha subunit isoform 1 (IL-15RA), and an expression cassette for secretable PD-L1 blocking peptide was conjugated to human IgG4 Fc (TF-Fc) were constructed into OV Herpes simplex virus type 1 (HSV-1), termed hVG161(VG161). The mVG161 was identical to VG161 apart from the substitution of mouse IL-12 for human IL-12 and the presence of a mouse-specific version of the PD-L1 blocker conjugated with mouse IgG1 Fc. In contrast, mVG160 was the backbone neither express mouse IL-12, IL-15, IL-15RA, nor the PD-L1 blocking peptide.

2.4 Real-time quantitative PCR (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Sigma-Aldrich). The reverse transcription reaction was performed using a SuperScript RT-PCR kit (Thermo Fisher Scientific), and RT-qPCR was performed using SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology) according to the manufacturer's protocol. mRNA levels were normalized to GAPDH level, and the results were performed with at least three independent experiments. The primer sequences are listed in Supporting Information: Table 1.

2.5 Enzyme-linked immunosorbent assays (ELISAs)

The levels of human IL-12p70, human IL-15/IL-15RA complex, or PD-L1 blocker in VG161-infected cell culture supernatants were quantified using the following ELISA kits: Human/Mouse IL-12p70 and IL-15 Human/Mouse ELISA kits were from Proteintech (Chicago, IL, USA), Human IgG4 ELISA kit was from Thermo Fisher Scientific and Mouse IgG1 ELISA kit was from Abcam.

Mice serum was analyzed for Interferon gamma (IFN-γ) and Tumor Necrosis Factor alpha (TNF-α) production using the IFN-γ and TNF-α Mouse DuoSet ELISA Kit (R&D Systems) according to the manufacturer's recommended procedures.

2.6 In vivo tumor models and lung metastasis model

All animal experiments were performed according to the proven procedures by the Institutional Animal Care and Use Committee at Zhejiang Laboratory Animal Center. BALB/c mice were implanted orthotopically with EMT-6 cells on both sides, then the right-side tumors were injected intratumorally with either PBS control or mVG161 for five consecutive days. The left side tumors were not treated. Subsequently, tumor size (length × width²/2) and mouse weight were recorded every 2 days. After 23 days of treatment, mice were killed and tumors were dissected and photographed. EMT6-Luc cells (50 × 10⁴) were subcutaneously injected into the second mammary fat pad to establish an orthotopic transplantation model. The BALB/c mice were randomly divided into two or five groups: Vehicle, PTX, mVG161, PTX + mVG161, and mVG161 + PTX. Mice received mVG161 for three consecutive days and PTX every other day for two injections as indicated. After 19 days, lung metastasis was analyzed by an IVIS-100 imaging system (Xenogen). Mice were killed, the metastatic lung tissues were either fixed in picric acid solution and nodules were counted 72 h later or fixed in 4% paraformaldehyde and were detected in paraffin-embedded sections by hematoxylin and eosin (H&E) staining. After 120 h, immunohistochemical analysis of CD3⁺ and CD8⁺ T cells were performed on sections of the metastatic tumors.

2.7 RNA-seq and analysis

Tumors were collected 120 h after virus injection, then the total RNA was isolated using TRIzol reagent (Invitrogen). Library construction and sequencing were performed on a BGISEQ-500 platform by Wuhan Genomic Institution (www.genomics.org.cn, BGI). All reads were mapped to the mm10 mouse genome, and the uniquely mapped reads were subjected to RNA-seq data analysis using the Hierarchical Indexing for Spliced Alignment of Transcripts (HISAT) system.¹³ The raw data have been uploaded to the public database, BioProject ID: PRJNA949807.

2.8 Flow cytometry analysis

Tumors were harvested from mice and single-cell suspensions were made by incubating minced tumor at room temperature for 1 h in enzyme digestion buffer containing type V collagenase, type IV DNase, and type V Hyaluronidase. Cells were washed and incubated for 30 min on ice in staining buffer (Thermo Fisher Scientific) with different combinations of the following antibodies: Fixable Viability Dye eFluor780, PE-conjugated anti-CD8, AF488-conjugated anti-CD107a, PerCP-Cy5-5-conjugated anti-Ly-6C antibodies, and PE-conjugated anti-CD11c (Thermo Fisher Scientific); BUV395-conjugated anti-CD49b (BD Biosciences); PE-Cy7-conjugated anti-CD3, FITC-conjugated anti-CD11b, PE-Cy7-conjugated anti-Ly-6G, APC-conjugated anti-Ly-6C, eF450 anti-MHCII antibodies, BV785-conjugated anti-CD8, BV421-conjugated anti-CD45, and BV510-conjugated anti-CD45 (BioLegend). For intracellular staining of IFN-γ and TNF-α, cells pre-stained with antibodies targeting surface proteins were incubated with ×1 fixation/permeabilization buffer and subsequently stained with APC-conjugated anti-IFN-γ and FITC-conjugated anti-TNF-α antibodies (BioLegend) in ×1 permeabilization buffer (Thermo Fisher Scientific). FACS analysis was performed on a BD Fortessa X20.

2.9 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

Mouse tumor tissues were fixed in 4% (w/v) paraformaldehyde (PFA), dehydrated in an ethanol gradient, and embedded in paraffin. Samples were then cut into 3 μm sections using a Leica microtome. TUNEL assays were carried out according to the manufacturer's instructions (Roche Ltd.). Sections were analyzed using an inverted fluorescence microscope (Olympus).

2.10 Immunohistochemistry (IHC)

Mouse tumor tissues were fixed in 4% PFA, dehydrated in an ethanol gradient, and embedded in paraffin. Samples were then cut into 3 μm sections using a Leica microtome. Staining for caspase-3 (Thermo Fisher Scientific) was carried out according to the manufacturer's instructions.

2.11 Immunofluorescence staining and microscopy

Mouse tumor sections were mounted on glass slides and deparaffinized. For double immunofluorescence staining, sections were incubated in 3% skim milk in PBS-T for 30 min and then incubated overnight at room temperature with a combination of two primary antibodies. These combinations included rat monoclonal anti-CD3 (Proteintech), anti-CD4 (Proteintech), and anti-CD8 (Proteintech) antibodies. Sections were then incubated with a mixture of fluorophore-labeled secondary antibodies and counterstained with Hoechst. Immunofluorescence analysis of CD3⁺ and CD8⁺ T cells were performed on sections of the metastatic tumors. The images were analyzed using an inverted fluorescence microscope (Olympus).

2.12 Measurement of biochemical parameters

Whole blood was collected from the retro-orbital veins of mice using heparinized capillary tubes. After centrifugation, sera were collected and the levels of serum blood urea nitrogen (BUN), creatinine (Cr), uric acid (UA), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), and alanine (ALT) were analyzed using the AU680 Chemistry System (Beckman Coulter).

2.13 Statistical analysis

Statistical analysis was performed with GraphPad Prism 8 (La Jolla). Data are expressed as the mean ± standard deviation (SD) and comparisons were made using a two-tailed Student's t-test or one-way analysis of variance. In all statistical tests, significance levels (p-values) are presented on the figures. *p < 0.05, **p < 0.01 and ***p < 0.001.

3.1 VG161 inhibits BC and lung metastasis

We first assessed the oncolytic capability of VG161 in BC cells. SK-BR-3, MDA-MB-231, MCF-7, and EMT-6 cells were infected with VG161 or mVG161 for 24, 48, and 72 h at MOIs ranging from 0.01 to 10. Cell viability was quantified by CCK-8 assays, which suggested that the VG161 virus inhibited viability in a dose-dependent manner (Figure 1A). Next, we detected the ability of VG161 to replicate in different tumor cell lines with plaque assays, and the result suggested that VG161 replicates effectively in the indicated cell lines (Figure 1B). In vivo experiments were used to further evaluate the efficacy of mVG161 after intratumoral inoculation in EMT6 tumor-bearing animals. BALB/c mice were implanted orthotopically with EMT-6 cells on both sides, then the right-side tumors were injected intratumorally with either PBS control or mVG161 for five consecutive days. The left side tumors were not treated. In animals treated with mVG161, the tumors were significantly regressed on the injected side with both 1 × 10⁵ and 1 × 10⁷ PFU virus. Interestingly, the non-injected side tumors were also regressed after 1 × 10⁷ PFU mVG161 virus treatment (Figure 1C). Previous work by Chouljenko et al demonstrated that VG161 could induce a potent antitumor immune response in colon cancer.⁸ We hypothesized that this could also be applied to BC and that VG161 may induce antitumor immune responses that can inhibit the nontreated side tumors. Additionally, VG161 treatment resulted in significantly reduced lung metastasis of the tumors (Figure 1D−E). These data confirm that VG161 can repress BC cell proliferation and lung metastasis.

3.2 VG161 enhances the immune response in BC

VG161 is a genetically modified HSV consisting of IL-12, IL-15, IL-15RA, and a fusion protein (TF-Fc) capable of blocking PD-1/PD-L1 interactions. We first confirmed that the expression of human IL-12/IL-15 and PD-L1 blocker (IgG4) (Figure 2A–C) or mouse IL-12/IL15 and mIgG1 (Supporting Information: Figure 4) were strongly increased in the supernatants of VG161-infected BC cells. To further evaluate the antitumor immune response induced by VG161, we also used a dual tumor EMT model. We examined various immunostimulatory pathways in EMT6 tumors taken from BALB/c mice that were treated with either mVG161 on the right side or untreated on the left side using high-through sequencing of cDNA libraries (RNA-seq). Several immunostimulatory pathways, such as the TNF-α, NF-κB, and IL-17 signaling pathways, were activated in both the treated and untreated tumors (Figure 2D). RT-qPCR analyzed the expression of relevant genes in TNF and IL-17 pathway and the result illustrated the increased levels of those genes (Figure 2E). mVG161-injected right side and uninjected left side tumor sections were also stained and analyzed with IHC. The results displayed enrichment of CD3, CD4, and CD8 expression in mVG161-injected tumors compared with vehicle groups, suggesting the presence of tumor-infiltrating lymphocytes (TILs) in the TME (Figure 2H). Moreover, the antitumor immunity signature elicited by mVG161 was robust, as seen by increased production of IFN-γ and TNF-α (Figure 2F). Additionally, there were no abnormal changes noted in the liver and renal functional tests (Figure 2G), and the hematoxylin and eosin (H&E) staining of liver, heart, spleen, lungs and kidneys of mice were showed no significant damage (Supporting Information: Figure 5), suggesting a good safety profile in vivo. These findings further support the conclusion that VG161 can stimulate an antitumor immune response in BC in addition to oncolytic activity, with no apparent toxicity in the liver and kidney.

3.3 VG161 and PTX synergistically demonstrate greater tumor repression and tumor killing in vivo

Oncolytic HSV treatment is commonly combined with chemotherapy. This combination approach can achieve more efficient cancer cell killing than using either agent alone.^14-16 To investigate this concept in our BC model, we combined mVG161 and PTX treatments and examined the antitumor efficiency in vivo and in vitro. Cells were pretreated with either VG161 or PTX for 24 h, followed by treatment with PTX or VG161 for 24, 48, or 72 h. CCK-8 assays showed that VG161 and PTX in combination could further suppress MCF-7 and MDA-MB-231 cell viabilities compared with either treatment alone. Notably, pretreatment with VG161 showed more significant effects than pretreatment with PTX (Figure 3A). We then confirmed our in vitro findings in tumor-bearing animals. EMT-6 cells were orthotopically implanted on both sides of BALB/c mice. Right side tumors were either injected intratumorally with mVG161 for three consecutive days followed by PTX treatment or pretreated with PTX followed by mVG161 infection. These results were consistent with our in vitro experiments showing that treating with mVG161 first had more efficient antitumor effects (Figure 3B). Next, to further verify whether the inserted genes carried by mVG161 can provide enhanced antitumor activity, we used mVG160 backbone virus as a positive control group. The result showed that PTX combined with mVG161 had stronger antitumor effect than PTX combined with mVG160, and the body weights of the mice in the six groups did not differ to a statistically significant extent (Figure 3C). In addition, we performed TUNEL assays and IHC analysis for the proapoptotic molecule caspase-3 in the right-side tumors. We found that the mVG161-PTX combination can induce greater apoptosis levels (Figure 3D–E). These results suggest that inducing an immune response with mVG161 before PTX treatment may enhance the effects of this chemotherapy to effectively fight BC.

3.4 VG161 infection with PTX cotreatment reduces metastatic pulmonary lesions in the EMT-6 BC model

BC is a highly invasive disease and often metastasizes to the lungs. EMT6-Luc BC cells that were orthotopically implanted into BALB/c mice effectively metastasized to the lung and formed metastatic tumors. The number of metastatic tumors in the lungs was significantly reduced after mVG161 infection, as seen by bioluminescence ex vivo imaging and H&E staining. Metastatic pulmonary lesions were further decreased after cotreatment with PTX (Figure 4A–B). Additionally, IHC analysis showed enhanced infiltration of CD3⁺ and CD8⁺ T cells in the metastatic pulmonary lesions (Figure 4C). These findings indicate that VG161 in combination with PTX can decrease BC pulmonary metastasis and provide potential new therapeutic options for treating metastatic BC.

3.5 VG161 infection with PTX cotreatment greater alters the TME

To determine the antitumor immune response of mVG161 infection with PTX cotreatment, we utilized flow cytometry to analyze cells isolated from excised EMT-6 tumors of immunocompetent BALB/c mice treated with mVG161, PTX, or their combination. Infiltration of lymphoid cells, including CD8⁺ T cells and natural killer cells (NKs), as well as IFN-γ, TNF-α, and CD107a expression levels in these cells, increased following mVG161 injection. These observations were further enhanced following cotreatment with PTX. Myeloid cell infiltration, including macrophages, myeloid-derived suppressor cells (m-MDSCs), and dendritic cells (DCs), were also increased after this cotreatment (Figure 5A–B). These data show that VG161 infection followed by cotreatment with PTX can profoundly alter the TME composition to support a robust antitumor immune response in BC.