Cell line and oncolytic virus
The CT26, MC38, 4T-1, and RAW264.7 cell lines were purchased from the National Infrastructure of Cell Line Resource (Beijing, China) and kept by our laboratory. The cells were cultured in a constant temperature incubator containing 5% CO2 at 37°C.
OH2 was provided by Binhui Biopharmaceutical Co., Ltd. (Wuhan, China). The virus was an attenuated OH2 derived from the wild-type HSV-2 strain HG52 deleted the ICP47 gene and ICP34.5 gene [18, 19].
Lysate preparation
The CT26, MC38, and 4T-1 cell lines in the logarithmic growth phase were passaged to 10 cm2 culture dishes when the cells reach 70–80% confluence, rinse the cells with PBS and then add 5 ml serum-free RPMI-1640 medium (HyClone, Waltham, MA) and infect the cells with OH2 according to MOI=1, and add 5 ml RPMI1640 medium containing 10% FBS (Gibco, Waltham, MA) after 1 h. After 30 h, the cell supernatant was collected, centrifuged at 4°C, 400 g for 5 min, and the lysate was collected and stored at −80°C for later use. The cell-free supernatant (CFS) of CT26, 4T-1, MC38, and untreated RAW264.7 were used as control. The CFS of CT26, 4T-1, and MC38 was obtained from the culture supernatant of cells in a logarithmic growth phase, centrifuged at 4°C, 400g for 5 min. The supernatant of CT26, MC38, and 4T1 cell lysate prepared by repeated freezing and thawing was also used as the control group.
Cell viability test
RAW264.7 cells in the logarithmic growth phase were spread on a 96-well plate at 2000 cells/well and cultured for more than 6 h. After the cells adhered, added the lysate and controls were added to the cells, and Cell Counting Kit-8 (CCK8) detection was performed at the corresponding time points (0 h, 6 h, 12 h, 24 h, 48 h) (Dojindo, Kumamoto, Japan). Before detection, 100 μl of detection working solution (CCK8 reagent: RPMI1640 medium = 1:10) was added to each well and incubated for 1 h in a 37°C, 5% CO2 incubator in the dark. Finally, a microplate reader (Bio-Rad, Japan) was used to detect the absorbance of the cells at a wavelength of 450 nm.
Flow cytometry to detect the polarization direction of macrophages
RAW264.7 cells in the logarithmic growth phase were spread to a 6-well plate at 106 cells/well. After at least 6 h, the cells were completely attached to the wall, and the lysate and control were added separately. After 24 h of treatment, RAW264.7 cells were stained with FITC anti-mouse F4/80 (clone: FJK-16s, Invitrogen, Waltham, Massachusetts), APC anti-mouse CD86 (clone: GL-1, Biolegend, San Diego, CA) and PE anti-mouse CD206 (clone: MR6F3, Invitrogen, Waltham, Massachusetts) according to the protocol of the antibodies (M1 macrophage: F4/80+CD86+, M2 macrophage: F4/80+CD206+) and subjected to flow cytometry (LSR II, BD). Detection of SIRPα on macrophages in spleen from mouse used FITC anti-mouse F4/80, APC anti-mouse CD11b (clone: M1/70, Biolegend, San Diego, CA), and PE anti-mouse SIRPα (clone: P84, Biolegend, San Diego, CA). All flow cytometry used to detect macrophage typing in this study was set up with an antibody isotype control group (PE Rat IgG2a, clone: RTK2758, Biolegend, USA; APC Rat IgG2a, clone: RTK2758, Biolegend, USA; FITC Rat IgG2a, clone: RTK2758, Biolegend, USA).
Functional experiments on macrophages
The CT26, MC38, and 4T-1 cell lines in the logarithmic growth phase were digested and resuspended at a concentration of 1×106/mL. One μL of CFSE (C34554, Life Technology, Waltham, MA) working solution (0.5 mM) was added to every 2ml of tumour cells with a concentration of 1×106/mL and incubated at 37°C (5% CO2) for 8 min. After the incubation, 10ml of pre-cooled RPMI 1640 medium containing 10% FBS was added to stop staining. The supernatant was discarded by centrifugation, and the cell concentration was adjusted to 5×105/mL with RPMI 1640 medium with 10% FBS. The concentration of raw264.7 treated with lysate and control was adjusted to 5×106/mL. With the ratio of 25:1, 50:1, and 100:1 as the effector to target ratio parallel samples, co-cultivation was carried out in a 96-well U-shaped well plate, 100μL raw264.7 cells, and 100μL tumour cells were added to each well, and 3 parallel samples were set up. Then placed the culture plate a 37°C (5% CO2) for 4 h. Before testing, 200 μL of PI (P8080, Solarbio, Beijing, China) working solution (2.5μg/mL) was added to each culture well.
Detection of the expression level of CD47 and SIRPα in vivo and in vitro
The CT26, MC38, and 4T-1 cell lines and macrophages in the spleen of mice were stained with APC anti-mouse CD47 (clone: miap301, Biolegend, San Diego, CA), purified anti-mouse SIRPα (clone: P84, Biolegend, San Diego, CA) and anti-mouse IgG-Alexa 488 (ab150113, Abcam, Cambridge, UK) antibodies according to the protocol of the antibodies. The expression levels of CD47 and SIRPα were detected by flow cytometry.
Immunohistochemical staining
The paraffin-embedded tissue sections were baked at 60–65°C for more than 6 h and then put in xylene for dewaxing while hot. Steps were followed by gradient ethanol hydration, citrate repair antigen (ZLI 9064, Zsjqbio, Beijing, China), 3% hydrogen peroxide blocking endogenous peroxidase activity, blocking (SP-KIT-B2, MXB, Fujian, China), primary antibody F480 (clone: SP115, dilution: 1:200, Abcam, Cambridge, UK), CD86 (clone: E5 W6H, dilution: 1:500, CST, Danvers, Massachusetts), CD206 (polyclonal, dilution: 1:100, Abcam, Cambridge, UK), CD8 (polyclonal, dilution: 1:200, Affinity Biosciences, Jiangsu, China), CD16 (polyclonal, dilution: 1:200, Affinity Biosciences, Jiangsu, China) and horseradish peroxidase (HRP)-conjugated secondary antibody (Kit-5010, MXB, Fujian, China) incubation, visualized with the 3,30-diaminobenzidine DAB (DAB-1031, MXB, Fujian, China) chromogen, haematoxylin staining (Solarbio, Beijing, China), hydrochloric acid ethanol differentiation, and ammonia water returning to blue. Finally, after dehydration with gradient ethanol, tissue sections were dehydrated in xylene and sealed with neutral gum. After drying, tissue sections were observed for staining under a microscope (Nikon Eclip se 80i, Japan).
Multicolour immunohistochemical staining
The paraffin-embedded tissue sections were baked at 60°C-65°C for more than 6 h and then put in xylene for dewaxing while hot. After gradient ethanol hydration and neutral formalin immersion, each stained antibody was sequentially repaired by citrate, blocked by goat serum, and incubated with primary antibodies against F480 (clone: SP115, dilution: 1:500, Abcam, Cambridge, UK), CD86 (clone: E5 W6H, dilution: 1:1000, CST, Danvers, Massachusetts), CD206 (polyclonal, dilution: 1:200, Abcam, Cambridge, UK), CD8 (polyclonal, dilution: 1:500, Affinity Biosciences, Jiangsu, China), CD16 (polyclonal, dilution: 1:500, Affinity Biosciences, Jiangsu, China) and secondary antibody, and fluorescently stained to amplify the signal. After the staining was completed, the DAPI working solution was added dropwise, and finally, the super anti-quenching mounting tablet was added to the mount as required by the instructions in the kit (TSA-RM, PANOVUE, Beijing, China). The stained tissue slices were scanned and analysed with Plaris and Inform software.
IHC images were scanned using CaseViewer2.4 software. The degree of staining was scored: cells with staining <10% were scored as negative staining (−, 1), cells with staining 10–49% were scored as (+, 2); cells with staining rate of 50–74% were scored as (++,3), 75–100% stained cells were denoted as (+++, 4). Staining positive range scores are as follows: colourless (0); pale yellow particles (1), tan particles (2), and brown particles (3). The final score was defined as the staining extent score multiplied by the staining positive range score [20]. Negative expression scores ranged from 0 to 5, with positive expression scores over 5 [21].
Animal model construction
The mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing Vital River Laboratory Animal Technology Co., Ltd.). Six- to 8-week-old female Balb/c mice weighing approximately 19–20 g were kept in a laminar flow ultraclean rack in our animal room under specific aseptic conditions for approximately 1 week. Each mouse was inoculated subcutaneously (s.c.) with 3×105 CT26 cells on the right side of the dorsal area. The tumour appeared in approximately 5–7 days, and treatment was given when the diameter of the tumour grew to 3–5 mm or 8–10 mm.
All operations were carried out under the standard operating procedures of the specific pathogen-free (SPF) experimental mouse breeding management system specified by the state. All animal-related experimental procedures were approved by the Committee on the Ethics of Animal Experiments of the National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences (CAMS), and Peking Union Medical College.
The first part
CT26 cell subcutaneous transplantation tumour model was constructed. When the diameter of the tumour reached 3–5 mm (day 7), the mice in the CL model were separated into three groups (OH2 [OH2 + Control Liposomes], OH2 + CL, and PBS control group) with an even distribution of tumour volumes. Assigned more than 10 mice to each group. OH2 (2×106 plaque-forming units [PFU]) was performed by intratumoural injection (i.t.) on day 0, day 2, and day 4 in a volume of 100 μl. One hundred microliters of CL (7 mg/mL, F70101C-NC, FormuMax, Sunnyvale, CA) or control liposomes (7 mg/mL, F70101-N, FormuMax, Sunnyvale, CA) per mouse was given by intraperitoneal injection (i.p.) at the day before the OH2 treatment. The PBS control group was given 100 μL PBS per mouse. Tumour growth was regularly observed and recorded for each group of mice. After 14 days of treatment, the tumour tissues were resected for immunohistochemical and multicolour immunohistochemical staining. The mice in the survival observation group were tumour-bearing again on the 40th day.
The second part
A CT26 cell subcutaneous transplantation tumour model was constructed. When the diameter of the tumour reached 3–5 mm (day 7), it was defined as the tumour being smaller at the time of initial treatment. The mice in the anti-SIRPα model with smaller tumours at initial treatment were separated into six groups (OH2 + anti-SIRPα antibody group, OH2+isotype group, OH2 group, anti-SIRPα antibody group, isotype group, and PBS control group) with an even distribution of tumour volumes. Assigned more than 10 mice to each group. The OH2 treatment or PBS was injected on days 0, day 2, and day 4, and the anti-SIRPα antibody (clone: P84, Bioxcell, Lebanon, New Hampshire) or isotype (TNP6A7, Bioxcell, Lebanon, New Hampshire) was administered on days 1 and 3. The anti-SIRPα antibody and isotype were injected intraperitoneally (i.p.) with 100 μg, diluted to 1 mg/ml with PBS without preservation solution, and injected 100 μL intraperitoneally for each mouse. The PBS control group was given 100 μL PBS per mouse. The administration method and dosage of the OH2 were the same as those in the CL model. The tumour growth of mice was observed and recorded regularly every two days.
When the diameter of the tumour reached 8–10 mm (day 14), it was defined as the tumour being larger at the time of initial treatment. The mice in the anti-SIRPα model with larger tumours at initial treatment were separated into four groups (OH2+ anti-SIRPα antibody group, OH2+isotype group, OH2 group, anti-SIRPα antibody group, and PBS control group) with an even distribution of tumour volumes. Assigned more than 10 mice to each group. The mode of administration and treatment strategy was the same as described above. The tumour growth of mice was observed and recorded regularly. After 12 days of treatment, the tumour tissues were resected for immunohistochemical, multicolour immunohistochemical staining, and RNA sequencing.
When the experimental endpoint or humanitarian endpoint was reached, such as when the size of the mouse reached 2500 mm3 or tumour metastasis or rapid growth caused ulceration, necrosis, or infection that interfered with eating or walking, the mice were anaesthetized with 5% chloral hydrate and then sacrificed by cervical dislocation. The calculation formula of tumour volume was volume = (length×width2)/2.
RNA sequencing
The transcriptome sequencing involved in this study was undertaken and completed by Tianjin Nuohe Zhiyuan Bioinformation Technology Co., Ltd. RNA samples were required to reach a total volume of ≥30 μL, a total volume of ≥1.5 μg, and a concentration of ≥50 ng/μL. The agarose gel electrophoresis quantification method, Nanodrop, and Agilent 2100 were used to test the concentration, purity, and integrity of the submitted RNA. The type of library construction was a eukaryotic chain-specific library. The sequencing strategy was Illumina Hiseq-PE150 (two-way sequencing).
Single-cell RNA sequencing
The tumour tissues of the control group, OH2 group, and combined treatment group were subjected to single-cell sequencing analysis, and the single-cell sequencing technology used in this study was provided by Huada Company. The tissue was dissociated using the Tumor Dissociation Kit (mouse, MACS), the dead cells were removed, and the single-cell suspension of living cells was left for sequencing. The data obtained were analysed using the Seurat package, and the volcano plot was drawn using the R software package EnhancedVolcano. The R packages used for GO analysis and KEGG analysis included tidyverse, patchwork, monocle, clusterProfiler, org.Mm.eg.db.
Bioinformatics analysis
R software version 4.0.2 was used to analyse the transcriptome sequencing data, and the R software package “edgeR” was used for differential expression analysis between groups. Gene set enrichment analysis (GSEA) was used to study the difference signal pathways between different groups. The R software package “clusterProfiler” was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Gene set variation analysis (GSVA) was used for immune cell infiltration analysis. Student’s t-test was used to compare the differentially expressed genes, and the P-value was adjusted and corrected by the Benjamini-Hochberg method. The log2-fold-change was greater than 1, and the corrected P-value was less than 0.01 as the threshold to judge the significance of the difference. A total of 770 immunology-related mouse genes created from the nCounter Mouse PanCancer Immune Profiling Panel (NanoString) were used as reference genes which are listed in Additional file 1: Table S1 and Additional file 2: Table S2.
Statistical analysis
GraphPad Prism software version 8 was used for statistical analysis and statistically significant differences were defined as a p value<0.05. Two-tailed, unpaired Student’s t-test was used to compare two groups. Two-way analysis of variance (ANOVA) was performed on the experimental data for tumour volumes for more than two groups. The Kaplan-Meier method and log-rank test were used for the survival curve data; the survival period was defined as the time from the start of treatment to the end of the observation. The results are presented as the mean ± S.E.M. In this study, all experiments were repeated at least three times, except for the parts otherwise stated.
