Patients and specimens
This study was approved by the Nanfang Hospital Ethics Review Board. A total of 51 paraffin-embedded samples from patients with colon cancer were collected. According to the medium tumour size, patients were divided into 26 LTB (average TB of 13.00 cm³) and 25 HTB (average TB of 88.11 cm³). Metastatic tumor needle biopsy samples of 2 patients with colon cancer liver metastasis were collected. The patients were all histologically diagnosed with colon cancer at Nanfang Hospital, Southern Medical University (Guangzhou, China). We also downloaded 1 colon cancer datasets with clinical information from TCGA (COAD) and 1 melanoma datasets with clinical information from TCGA (SKCM)(https://portal.gdc.cancer.gov/).
Cell culture
MC38 colon cancer cell line was grown in 1640 RPMI (Gibco) supplemented with 10% FBS (Hyclone), 2 mM glutamine and 20 μg/ml gentamicin. B16 melanoma cell line was grown in high-glucose DMEM (Hyclone) supplemented with 10% FBS (Hyclone), 2 mM glutamine and 20 μg/ml gentamicin. RAW 264.7 cell line was grown in high-glucose DMEM (Hyclone) supplemented with 10% FBS (Hyclone), 2mM glutamine and 20μg/ml gentamicin. All cells were grown at 37 ℃ in a humidified atmosphere with 5% CO2. All cell lines were authenticated by the STR (Short Tandem Repeat) profiling.
Mice
C57BL/6J female mice (3-4 weeks old) from Southern Medical University Laboratory Animal Center were housed at 25 ℃, and humidity was 50–60%. The light cycle consisted of light for 12 hours and dark for 12 hours.
Reagents
Anti-PD-1 antibody (10 mg/kg, BioXCell, Cat.No. BE0146), PLX3397 (40 mg/kg, Selleck, Cat.No. S7818), anti-PD-L1 (5 μg/ml, BioXCell, Cat.No. BE0101), Anti-Mouse CD45 APC (MultiSciences, Cat.No. AM04505-100), Anti-Mouse CD3 eFluor® 506 (eBioscience, Cat.No. 69-0032-82), Anti-Mouse CD8a Alexa Fluor®700 (eBioscience, Cat.No. 56-0081-82), Anti-Mouse CD11b FITC (MultiSciences, Cat.No. AH011B01-100), Anti-Mouse F4/80 PE (MultiSciences, Cat.No. AM048004-100), Anti-Mouse CD11c APC (MultiSciences, Cat.No. AM011C05-100), Anti-Mouse CD206 PE-Cyanine7 (MultiSciences, Cat.No. 25-2061-80), Anti-Mouse PD-L1 PerCP-Cy5.5 (MultiSciences, Cat.No. AM27407-20), Anti-Mouse IFN-γ APC (MultiSciences, Cat.No. AM0IF05-100), CD8 mouse monoclonal antibody (Proteintech, Cat.No. 66868-1), CD8 rabbit monoclonal antibody against mouse (Abcam, Cat.No. ab217344), ANNEXIN V-FITC/PI apoptosis detection Kit (Solarbio, Cat.No. CA1020), CFSE (Targetmol, Cat.No. T6802), Anti-Mouse Ki-67 PE (MultiSciences, Cat.No. 12-5698-80), Recombinant Mouse IL-2 (Solarbio, Cat.No. P00198), Mouse CD3 / CD28 (ThermoFisher, Cat.No. 11456D), IGFBP2 ELISA (Jiangsu Yutong, Cat.No. F5217-A), Mouse cytokine antibody array C1 (RayBiotech, Cat.No. AAM-CYT-1000-2), FLLL32 (Selleck, Cat.No. 1226895-15-3), HJC0152 (Selleck, Cat.No. S8561).
Mice model construction
① For LTB and HTB mice model construction. MC38 colon cancer cells or B16 melanoma cells were used to construct LTB (0.5×106 cells, subcutaneous growth for 14 days) and HTB (2.0×106 cells, subcutaneous growth for 14 days) mice models. Mice were intraperitoneally injected with physiological saline or anti-PD-1 antibody on day 14, day 17, day 20 and day 23. Mice were sacrificed on day 26. ② For various TB mice model construction. MC38 colon cancer cells or B16 melanoma cells were used to construct LTB (0.5×106 cells, subcutaneous growth for 14 days), HTB (2.0×106 cells, subcutaneous growth for 14 days), SS3w (0.5×106 cells, subcutaneous growth for 21 days), TS1 & TS2 (TS1: 0.5×106 cells, subcutaneous growth for 14 days at left side. TS2: 0.5×106 cells, subcutaneous growth for 14 days at right side), TS2w & TS3w (TS2w: 0.5×106 cells, subcutaneous growth for 14 days at left side. TS3w: 0.5×106 cells, subcutaneous growth for 21 days at right side) mice models. Mice were sacrificed on day 21. ③ For surgery animal model construction. MC38 colon cancer cells were injected subcutaneously on the left (2.0×106 cells) and right (0.5×106 cells) sides of the same mouse. On day 14, surgery was operated on the left subcutaneous tumor to make TB the same as that on the right side. On day 16, 18 and 21, CD3+CD8+ T cells of subcutaneous tumor were dected by flow cytometry. ④ For drug combination mice model construction. 2.0×106 MC38 colon cancer cells or B16 melanoma cells were injected subcutaneously. Mice were divided into 4 groups: NC group, PLX3397(day 7)+anti-PD-1 group, PLX3397(day 13)+anti-PD-1 group and anti-PD-1 group. NC group without any treatment. PLX3397(day 7)+anti-PD-1 group was treated with PLX3397 on day 7, 9, 11, PLX3397(day 13)+anti-PD-1 group under the same treatment on day 13, 15, 17. PLX3397(day 7)+anti-PD-1 group and PLX3397 (day 13)+anti-PD-1 group were treated with anti-PD-1 on day 19, 22, 25, 28. Anti-PD-1 group just under anti-PD-1 treatment on day 19, 22, 25, 28. Mice were sacrificed on day 31 for dected CD3+CD8+ T cells of subcutaneous tumor.
Immunohistochemistry (IHC) staining
IHC was performed on 4 mm sections of formalin-fixed paraffin-embedded (FFPE) tissues. Paraffin sections were baked at 65 ℃ for 4 hours. Sections were deparaffinized, dehydrated through a graded ethanol series. Then sections were repaired in 0.01 M and pH 6.0 citrate buffer. Endogenous peroxidase was blocked by incubation in 0.3% H2O2 for 10 min. After incubating with 5% BSA for 1 hour at room temperature, sections were incubated with appropriate primary antibodies overnight at 4 ℃. Then sections were incubated with relevant secondary antibody for 1 hour at 37 ℃. Sections were incubate with a DAB substrate kit. CD8 protein IOD were calculated by image pro plus 6.0.
Preparation of LTCS and HTCS and supernatant-conditioned macrophages
LTB-tumor tissue culture supernatants (LTCS) or HTB-tumor tissue culture supernatants (HTCS) were prepared by plating tumor tissues in 1 mL DMEM for 24 hours.The supernatant was then centrifuged and harvested. To generate supernatant-conditioned macrophages, RAW 264.7 were first harvested and cultured with 50% LTCS or HTCS for 24 hours, and then washed with DMEM for three times. RAW 264.7 cultured with DMEM were used as controls.
Macrophage survival assay. RAW 264.7 were stimulated with 50% LTCS or 50% HTCS, or NC for 24 hours, and then were harvested. Macrophage survival was quantifified using ANNEXIN V-FITC/PI apoptosis detection Kit (Solarbio, Cat.No. CA1020).
In vitro macrophage-T cell co-culture system. In a 5-day incubation, bead-purified peripheral CD8+ T cells (1×105 cells/well in 96-well plates) were labelled with CFSE and co-cultured with macrophages (NC) or LTB MC38 subcutaneous tumour-infiltrating macrophages or HTB MC38 subcutaneous tumour-infiltrating macrophages or LTCS-conditioned or HTCS-conditioned RAW 264.7 in 200 μL RPMI-1640 medium containing rhIL-2 (20 IU/mL), anti-CD3/CD28 (2 μg/mL) antibodies, 2:1 for 5 days with or without anti-PD-L1 (5 μg/ml).
Flow cytometry
Cells were resuspended to achieve a cell concentration of 1×107/ mL. 100 μL cells were incubated with appropriate primary antibodies at 4 ℃ for 30 min. Cells were repeatedly washed twice using PBS, then cells were detected by flow cytometer. Raw data were analysed through the FlowJo_V10 software.
RNA-seq analysis of MC38 subcutaneous tumour cells
Total RNA in the tissue samples was extracted using Trizol. The mRNA was isolated and purified from the total RNA by using the Oligo (dT) magnetic beads. First-strand Illumina-barcoded libraries were generated using the NEB RNA Ultra Directional kit according to the manufacturer’s instructions. Sequencing was performed by a Illumina Hiseq sequencer. Data were aligned to mouse reference genome mm10 using Bowtie2. Normalized counts and differential expression analysis was performed using the DESeq2 R package. Gene enrichment analysis was performed using the R language clusterProfiler analysis package.
Single-cell RNA sequencing
Fresh B16 melanoma subcutaneous tumor cells were washed and resuspended in 1x PBS (calcium and magnesium free) containing 0.04% BSA. 10,000 living cells were loaded on a Chromium Single Cell Controller to generate Gel Bead-In-EMulsions containing all cDNAs. Sample demultiplexing, barcode processing, alignment, filtering, UMI counting, and aggregation of sequencing runs were performed using the Cell Ranger analysis pipeline (v1.2). Seurat R package was used to merge the scRNA-seq and cell clustering and calculate the principal components analysis (PCA). Cells in which fewer than 200 genes were detected and in which mitochondrially encoded transcripts constituted more than 10% of the total library were excluded from downstream analysis. Genes detected in fewer than 3 cells across the data set were also excluded. Significant PCs were chosen through the jackstraw method. The t-SNE dimensionality reduction was generated using the RunTSNE function and the UMAP was performed with the RunUMAP function. Unsupervised clustering using a shared nearest neighbour modularity optimization based algorithm (resolution parameter 0.8) identified 16 distinct clusters.
Mouse cytokine antibody array
Cytokines from LTB and HTB mouse melanoma were detected by AAM-CYT-1000-2 mouse cytokine antibody array. The original data obtained from the scans were background removed and normalized between chips using Raybiotech software. Differential proteins were screened using fold change (expression difference multiple) under the following selection conditions: ① fold Change ≤ 0.83 or fold Change ≥ 1.2; ② mean signal value per group > 150 .
ELISA
Subcutaneous tumors were grinded by the homogenate machine and centrifuged for 20 min at 2000 rpm. The supernatant was collected and then tested by the ELISA kit to determine the OD at 450 nm wavelength.
Statistical analyses
Graphpad Prism software (version 8.0.1) and SPSS software (version 26.0) were used for all statistical analyses. Quantitative data are shown as mean ± s.d. Statistical significance was determined using paired two-tailed Student’s t-tests. Values of p < 0.05 were considered significant. NSp > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001.