Cell culture

The 4T1 cells (CRL-2539, a mouse triple-negative mammary tumor cell line isolated from Balb/c mice), MDA-MB231 cells (HTB-26™, a human triple-negative mammary tumor cell line), B16F10 cells (CRL-6475, a murine melanoma cell line isolated from C57BL/6 J mice), RAW 264.7 cells (TIB-71, macrophage-like cell line derived from Balb/c mice), and HeLa cells (CCL-2™, human cervical cancer cells) were purchased from the American Type Culture Collection. The 4T1, RAW 264.7, and B16F10 cells were cultured in Roswell Park Memorial Institute (RPMI) containing 10% FBS and 1% antibiotics (streptomycin, 100 μg/mL; penicillin, 100 U/mL). Hela cells and MDA-MB231 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1% antibiotics (streptomycin, 100 μg/mL; penicillin, 100 U/mL).

Generation of recombinant CVB3

The miR-CVB3 was constructed as described previously [23]. In brief, 4 copies of miRNA-145 target sequence (TS), 4 copies of miRNA-216 TS, 2 copies of miRNA-1 TS, and 2 copies of miRNA-143 TS were inserted into the 5′untranslated region (UTR) of CVB3 genome. The resultant miR-CVB3 was propagated in Hela cells and kept at − 80 °C for further applications.

Preparation and characterization of CpGMel

A fixed concentration (10 μg/ml) of CpG oligodeoxynucleotides (CpG ODNs, 1826, Integrated DNA Technologies) was added to increasing concentrations (0, 3, 6, 12, 25, 40 μg/ml) of melittin (> 85% in purity, M2272, Sigma-Aldrich) and the mixture was incubated at room temperature for 1 h. The formation of CpGMel complex was evaluated using gel retardation assay (2.5% agarose gel). Moreover, 2.5% agarose gel was applied to evaluate the probability of binding CpGMel to the surface of miR-CVB3 after incubating them at room temperature for 1 h and purifying miR-CVB3 using a centrifugal filter.

Cellular uptake of miR-CVB3 and CpGMel

To evaluate the internalization/replication of miR-CVB3, 4T1 cells were seeded into the 8-well chamber slides (104 cells per well) and 24-well plates (5 × 104 cells per well). MDA-MB231 cells were also seeded into 24-well plates (5 × 104 cells per well). The following day, cells were exposed to miR-CVB3 (multiplicity of infection (MOI) = 1), or miR-CVB3 + CpGMel (miR-CVB3 at an MOI of 1, melittin at a concentration of 10 μg/ml, and CpG ODNs at a dose of 5 μg/ml) for 1 h. Then, the media was removed and replaced with fresh media. For examination of viral internalization/replication by confocal microscopy, after additional 16-h incubation, cells were washed with phosphate-buffered saline (PBS). After fixation in 4% paraformaldehyde and permeabilization with 0.1% Triton X-100, cells were blocked with 3% bovine serum albumin (BSA) and then incubated with VP1 antibody (M47, Mediagnost, Germany) at 4 °C for overnight. Following additional incubation with Alexa Fluor® 488-conjugated secondary antibody (A11029, Invitrogen) at room temperature for 1 h, cells were washed with PBS, mounted with fluoroshield with 4, 6-diamidino-2-phenylindole (DAPI; F6057, Sigma-Aldrich), and subjected to Zeiss LSM 880 inverted confocal microscopy for imaging.

For measurement of viral entry/replication by western blotting, after additional 16-h incubation, both 4T1 and MDA-MB231 cells were lysed in buffer (10 mm HEPES pH 7.4, 50 mm Na pyrophosphate, 50 mm NaF, 50 mm NaCl, 5 mm EDTA, 5 mm EGTA, 100 μm Na3VO4, and 0.1% Triton X-100). Western blotting was conducted using VP1 antibody as previously described [23].

To evaluate the impact of miR-CVB3 on internalization of CpGMel, CpGMel was prepared using CpG(Cy5) (CpG ODNs, 1826, Integrated DNA Technologies). Cells were seeded and treated with CpG(Cy5), CpG(Cy5)Mel, or miR-CVB3 + CpG(Cy5)Mel (concentration of CpG(Cy5) for all treatments was 5 μg/ml) for 5 h. Confocal microscopy and flow cytometry (Gallios Flow Cytometer) were applied to investigate the uptake of CpG(Cy5). The results of flow cytometry were analyzed with FlowJo version 10 software.

In vitro anti-cancer study Cell viability assay

The 4T1 and MDA-MB231 cells were seeded onto a 96-well plate (104 cells per well). The following day, cells were treated with miR-CVB3, CpGMel, or miR-CVB3 + CpGMel, as described above, for 24 and 48 h. Subsequently, 10 μl of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) solution (G9243, Promega) was added into the culture, followed by another 3-h incubation. The absorbance of each solution was measured using a microplate reader (BioTek Synergy H1) at 490 nm. The OD values of untreated cells were set as 100% viability, and the percentage of inhibition was then calculated.

Apoptosis detection

The annexin V-Fluorescein isothiocyanate (FITC) staining was exploited to assess the apoptosis of cells treated with miR-CVB3, CpGMel, or miR-CVB3 + CpGMel. In brief, after exposure to miR-CVB3, CpGMel, or miR-CVB3 + CpGMel for 24 h, 4T1 cells were harvested and resuspended in annexin binding buffer (V13246, Thermofisher Scientific). Subsequently, annexin V-FITC (5 μl) (A13199, Thermofisher Scientific) reagent was introduced to each sample and incubated for 20 min in the dark. Finally, the stained cells were analyzed using flow cytometry. Data were analyzed with FlowJo version 10 software.

Detection of danger-associated molecular patterns (DAMPs)

Release and cell surface exposure of DAMPs, including ATP, CRT, and HMGB1, were analyzed after 4T1 cells were exposed to single treatment (miR-CVB3 or CpGMel) or combination therapy (miR-CVB3 + CpGMel) for 24 and 48 h. Specifically, the release of ATP into the supernatant was measured using RealTime-Glo™ extracellular ATP assay kit (GA5010, Promega) according to the manufacturer’s protocol. Briefly, 20 μl of 4 × RealTime-Glo™ extracellular ATP assay reagent was added into each culture. The luminescence was then measured using a microplate reader (BioTek Synergy H1). HMGB1 release was assessed by western blotting using HMGB1 primary antibody (651401, Biolegend). Briefly, the supernatant after treatment was collected and precipitated by the addition of equal volume of methanol and 0.25 volumes of chloroform. The mixture was vortexed and centrifuged for 10 min at 20,000 × g. The upper phase was discarded. Subsequently, 500 μl was added into interphase. Afterwards, the mixture was centrifuged for 10 min at 20,000 × g. Finally, protein pellet was dried at 55 °C, resuspended in protein loading buffer, and subjected to western blotting. For CRT detection, after treatment, cells were incubated for 60 min with anti-CRT (Alexa Fluor® 647) antibodies (ab196159, Abcam) at 4 °C in the dark. Following several washes, flow cytometry was applied to analyze the translocation of CRT. Confocal microscopy was also used to visualize CRT on the surface of the cells exposed to corresponding treatments. After fixation, the cells were incubated with anti-CRT (Alexa Fluor® 647) for 1 h, followed by DAPI straining.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

RT-qPCR was conducted to measure the gene level of TNF-α and IL-6 in 4T1 cells treated with CpGMel, miR-CVB3, and miR-CVB3 + CpGMel for 8 h. Primers for the RT-qPCR analysis were synthesized by Integrated DNA Technologies and presented in Table 1. Briefly, after incubation, total RNA was isolated using RNeasy Mini Kit (Qiagen, 74104, Qiagen). The qPCR reaction containing 1 μg of RNA was conducted applying the TaqMan™ RNA-to-CT™ 1-Step Kit (4392653, Thermo Fisher Scientific) on a ViiA 7 Real-Time PCR System (Applied Biosystems). The results were normalized to β-actin mRNA. Samples were run in triplicate and analyzed using comparative CT (2 − ΔΔCT) method with control samples and presented as relative fold changes.

Table 1 Designed primers for RT-qPCRMacrophage activation

To examine the activation of macrophages in vitro, the media of 4T1 cells treated with miR-CVB3, CpGMel, or miR-CVB3 + CpGMel for 12 h were transferred to the plates seeded with RAW 264.7 cells, followed by incubation for 24 h. Subsequently, RAW 264.7 cells were collected and stained with CD80-PE (B340153, Biolegend) and MHC-II-Alexa Fluor® 647 (B346505, Biolegend) antibodies for 30 min. The macrophage activation was then detected using a flow cytometer. Data were analyzed with FlowJo version 10 software.

In vivo anti-tumor study Animals

Six- to 8-week-old female Balb/c (000651, The Jackson Laboratory) and female C57BL/6 J (000664, The Jackson Laboratory) mice were used for the in vivo studies. All animal procedures were performed in compliance with strict guidelines for the care and use of laboratory animals and were approved by the Animal Care Committee at the University of British Columbia (A18-0275). The ARRIVE guidelines were used for reporting animal research [37].

Therapeutic effects in a murine breast cancer model

The 4T1 cells (5 × 105 cells) in 100 μl of cold PBS were subcutaneously injected into the right flank of female Balb/c mice. After about 10 days, once tumor reached a palpable size (~ 50 mm3), 4T1 tumor-bearing Balb/c mice were randomly divided into 4 groups (n = 8 for each group), which were intratumorally treated with PBS, miR-CVB3 (105 Plaque-Forming Unit (pfu)/mouse), CpGMel (CpG = 50 μg/mouse and melittin = 100 μg/mouse), or miR-CVB3 + CpGMel, respectively. Treatments were performed twice on days 0 and 5. The length and width of the tumors were measured every 3 days using a digital caliper, and tumor volumes were calculated using the formula of (volume = length × width2 × 0.52). Furthermore, the tumor suppression rate (TSR) was calculated using the following formula: TSR (%) = [1 − (tumor volume of the treated group)/(tumor volume of the control group)] × 100 (%). According to our approved protocol, humane endpoints were defined as follows: mice losing ≥ 20% of their initial body weight, observation of ulceration in ≥ 10% of the tumor region, the tumor size reaching ≥ 1.7 cm in diameter, or tumor weight exceeding 10% of body weight. Mice were kept for 40 days to evaluate the survival rate. Mice in each group were euthanized once they reached humane endpoints. Additionally, tumor metastasis into the lungs was assessed at the end of the experiment. Briefly, lung tissues were collected and fixed in 4% paraformaldehyde. Metastatic tumors in the lung were analyzed via hematoxylin–eosin (H&E) staining. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was also performed on tumor tissues (collected at humane endpoints) to assess apoptosis according to the manufacturer’s protocol (G3250, Promega).

Safety analysis

To assess the safety of each treatment, the body weight of mice in each group was measured every 3 days until the experimental endpoint. For safety measurement, a different cohort of mice (n = 4 for each group) were treated with PBS, miR-CVB3, CpGMel, or miR-CVB3 + CpGMel as above. At 14 days post-treatment, mice were sacrificed. The heart, liver, spleen, lung, pancreas, and kidney were collected and fixed in 4% paraformaldehyde for H&E staining. In addition, facial blood was collected on day 14 for the blood biochemistry analysis for alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine (CREA), lipase (Lip), and cardiac troponin I levels by Advia 1800 (Advia 1).

Immune cell infiltration

Two weeks after treatment, mice were sacrificed, and tumor samples were paraffin-embedded and then sliced into 5 μm of thicknesses. The sections were deparaffinized, rehydrated, and then stained with anti-CD8 (sc-1177, Santa Cruz Biotechnology), anti-NK1.1 (14–5941-82, eBioscience), and anti-F4/80 (sc-377009, Santa Cruz Biotechnology) antibodies through immunohistochemistry (IHC), as previously described [38], using the MACH4 Universal HRP-Polymer Detection System (BRI4012H, Biocare Medical) and hematoxylin solution Gill II (GHS232, Sigma-Aldrich). Lastly, Aperio ScanScope AT (Digital slide scanner, Leica Biosystems Inc) was applied to attain whole-slide digital images. All the staining images were quantified using NIH ImageJ (version 1.52p) and the results were presented as relative optical density. Moreover, the level of IFN-γ, IL-6, and TNF-α, as well as translocated CRT and granzyme B, in tumor tissues was analyzed using immunofluorescence staining. Tumor tissues were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 10% FBS, and incubated with target antibodies including anti-IFN-γ (505802, Biolegend), anti-IL-6 (504502, Biolegend), anti-TNF-α (506302, Biolegend), and anti-CRT antibodies overnight. The following day, tissues were stained with Alexa Fluor® 488-conjugated secondary antibody (A11029, Invitrogen) for 1 h, followed by mounting with DAPI. Zeiss LSM 880 inverted confocal microscopy was used to visualize proteins.

Inhibition of distant metastatic tumor

For the distant tumor model, at 4 days after transplanting 4T1 cells into the right flank of mice (primary tumor, n = 5), a distant tumor was implanted by subcutaneous injection of 4T1 cells (5 × 105 cells) into the left flank of each mouse. The primary tumors were treated as described before. The length and width of the distant tumors were measured every three days using a digital caliper.

Therapeutic effects in a murine melanoma model

To evaluate anti-tumor activity of miR-CVB3 + CpGMel in melanoma tumor-bearing mice, B16F10 (5 × 105 cells) cells in 100 μl of cold PBS were subcutaneously injected into the right flank of C57BL/6 J female mice. After 10 days, once the tumor reached a palpable size (~ 50 mm3), the mice were randomly divided into 4 groups (n = 3 for each group). Mice were then intratumorally treated with PBS, miR-CVB3, CpGMel, or miR-CVB3 + CpGMel, and tumor size was measured as described above. At the experimental endpoint, various mouse organs were harvested for H&E staining and tumor was collected for viral quantitation by immunostaining of viral capsid protein VP1. We also assessed the expression of the coxsackievirus and adenovirus receptor (CAR) in non-treated implanted tumors (n = 3 mice) by IHC using an anti-CAR antibody (A1822, ABclonal).

Statistical analysis

Statistical analysis was conducted by GraphPad Prism V8.0.1 software and all data are expressed as mean ± standard deviation (SD) (n ≥ 3). The results were analyzed by unpaired Student’s t test or one-way ANOVA followed by Tukey’s test to determine differences. The differences between survival rates were assessed by log-rank test. P-value < 0.05 was considered to be statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001).