Antibodies, small interfering RNA (siRNA) and primer sequences
The primary antibodies used in the study are summarized in Additional file 5: Table S1. siRNA and primer sequences used in the study are summarized in Additional file 6: Table S2 and Additional file 7: Table S3.
Cell lines and cell culture
The CRC cell lines LS174T (CL-188), RKO (CRL-2577), DLD-1 (CCL-221), Caco2 (HTB-37), SW620 (CCL-227), HCT-8 (CCL-244), HCT116 (CCL-247), HCT-15 (CCL-225), normal colorectal epithelia cell line FHC (CRL-1831) and normal colorectal fibroblast cell line CCD-18Co (CRL-1459) were purchased from the American Type Culture Collection (ATCC). All CRC cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA). FHC was cultured in RPMI 1640 medium (Gibco, USA) supplemented with 15% fetal bovine serum (FBS) (Gibco, USA). CCD-18Co was cultured in EMEM medium (ATCC, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA). They were all cultured at 37°C in a humidified atmosphere with 5% CO2. Cell line certificates of analysis came from ATCC. All the cell lines were negative for mycoplasma.
Primary normal colorectal fibroblasts extraction and culture
Fresh normal colorectal mucosae were cut up with surgical scissors and then they were dissociated enzymatically in type IV collagenase (2.0 mg/ml, Sigma, C5138), hyaluronidase (0.4mg/ml, Sigma, H1115000) and DNase (25U/ml, Solarbio, D8071) in a 37 ℃ constant temperature shaker for 2h. After 2h, the tissues were passed through 40-µm cell strainer to generate single-cell suspensions. Centrifuge the single cell suspensions at 1200 rpm/min for 12 min, discard the supernatant, and resuspend the pellet with medium. Primary cells were then plated at a density of 1 × 105 viable cells in 25-cm2 adherent flasks in EMEM medium (ATCC, USA) with 10% FBS (Gibco, USA) in 5% CO2 at 37°C (trypsin digestion method). The tissues couldn’t pass through the strainer were transferred to the 25-cm2 adherent flasks. Add EMEM medium (ATCC, USA) with 10% FBS (Gibco, USA) into the adherent flasks after 24h when the tissue stuck to the bottom of the adherent flask and waiting for fibroblasts to crawl out of the tissues (improved tissue planting method).
CRC clinical specimens were obtained from patients who were pathologically diagnosed with CRC at Shenzhen Hospital, Southern Medical University. The study was approved by the ethics committee of Shenzhen Hospital, Southern Medical University, China.
IHC was performed on paraffin-embedded sections of CRC tissue according to standard LSAB protocol (Dako) using primary antibodies against α-SMA, CD90, FAP, CCL5 and CD31. The degree of staining in the sections was observed and scored independently by three pathologists. The percent positivity of CCL5 staining was scored from 0 to 4: 0 (0-5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). The staining intensity was scored on a 4-point scale: 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown), and 3 (strong staining, brown). Subsequently, the CCL5 expression score was calculated through multiplying the percent positivity score and staining intensity score. Accordingly, the expression level of CCL5 was defined as Low (0–4), Medium (5–8), and High (9–12).
Co-culture recruitment assay
Boyden Transwell chambers (Corning, 353097) were used following the instructions of the manufacturer. Briefly, 2 × 104 fibroblasts were incubated into the upper chambers. 1× 105 tumor cells or different concentration of CCL5 cytokine (Peprotech, 300-06-20) were incubated into the lower chamber. After 48h incubation, cells that successfully migrated were fixed with 4% paraformaldehyde, stained with Hematoxylin and counted in 5 random visual fields using a light microscope.
CCR1 inhibitor or CCR5 inhibitor treatment
Before the recruitment assay, fibroblasts were pre-cultured with CCR1 inhibitor (BX471, 100nM, MCE, ZK-811752) or CCR5 inhibitor (Maraviroc, 100nM, Selleck, UK427857) for 48h. During recruitment assay, BX471 or Maraviroc was put in both of the upper and the lower chambers.
siRNA of CCL5, CCR1, CCR3, CCR4, CCR5, CD44, GPR75 and SLC25A24 were purchased from GenePharma (Shanghai, China). HCT-8, SW620, CCD-18Co and primary normal colorectal fibroblasts were transiently transfected with siRNA using Lipofectamine 3000 Transfection Reagent (Invirtrogen, USA) in line with the manufacturer’s instructions.
Construction of stable cell lines
Lentivirus vector carrying the luciferase gene (Luc) and the human CCL5 sequence were obtained from GenePharma (Shanghai, China). An empty vector was used as control to CCL5 overexpression. According to the manufacturer’s instructions, stable cell lines were established by transfection of LS174T and RKO with these lentivirus vectors.
Enzyme linked immunosorbent assay (ELISA)
CCL5 levels in the conditioned medium (CM) of FHC and CRC tumor cells were measured by ELISA using a commercially available kit (Huamei Biotech, China), as described by the manufacturer’s instructions. The results were expressed in pg/ml, and the standard curve is based on the measured OD value of the standard product.
RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total cellular mRNA was extracted using TRIzol (TaKaRa). Prime-Script RT Reagent Kit with gDNA Eraser (TaKaRa) was used to reverse-transcribed mRNA into cDNA. And SYBR Premix Ex Taq (TaKaRa) was used for qRT-PCR.
Total proteins were isolated from cells using RIPA lysis buffer (FDbio, FD008),PMSF (FDbio, FD0100), Protease inhibitors (FDbio, FD1001) and protein phosphatase inhibitors (FDbio, FD1002). The concentration was determined using BCA protein assay kits (FDbio, FD2001). Total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% skimmed milk or 5% BSA for 1h at room temperature and then incubated with primary antibodies at 4°C overnight. Subsequently, the membranes were incubated with goat anti-rabbit or anti-mouse secondary antibody (FDbio, FDR007 and FDM007). The protein was detected by ECL chemiluminescence solution (FDbio, FD8030) and visualized using chemiluminescence detection system (Universal Hood II, Bio-Rad). The intensity of the immunoblot bands was quantified with the NIH ImageJ software.
Immunofluorescence of cells and CRC tissue
Immunofluorescence of cells was performed, as previously described . The steps of immunofluorescence of CRC tissue before incubation of primary antibody were the same as IHC. The following steps after incubation of primary antibody were the same as those of immunofluorescence of cells. Images were captured using the fluorescence inverted microscope.
Orthotopic xenograft colorectal cancer mouse model
BALB/C-nude mice (male, 3-5 weeks old) were purchased from the Gem Pharmatech Co., Ltd, Guangdong, China. All animal experiments were approved by the ethics committee of Shenzhen Hospital, Southern Medical University, China. 5X106 HCT116 cells were injected subcutaneously into the back of nude mice (n=3). After subcutaneous tumor formation, one of the tumor tissues was fixed and stained with hematoxylin-eosin (H&E), and the remaining tumor tissues were peeled off and cut into 1mm3 tumor tissue pieces with ophthalmic scissors. Choose 5 pieces from the cut tissue pieces and bury them in the cecal serosal layer of nude mice by purse string suture (n=5). After 8 weeks, the ceca of nude mice were surgically removed after euthanasia, fixed in 10% formalin, embedded in paraffin, and prepared into 2.5µm sections for H&E staining.
Human cytokine array
Serum-free CMs from FHC and CRC tumor cells HCT-8, HCT116, HCT-15, SW620 were collected after incubation for 24h and filtered through a 0.22-µm mesh. The CM samples were added to antibody arrays against 1000 unique cytokines (RayBio, GSH-CAA-X00) and processed according to the manufacturer’s protocol.
Total mRNA was extracted from fibroblasts before and after CCL5 stimulation. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions.
Sirius Red staining
Sirius Red staining was performed on paraffin-embedded sections of CRC tissue using Sirius Red Staining Kit (LEAGENE, DC0041), as described by the manufacturer’s protocol. The degree of staining in the sections was observed and scored independently by three pathologists under a polarized light microscope. The percent positivity of Sirius Red staining was scored from 0 to 4: 0 (0-5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). The staining intensity was scored on a 4-point scale: 0 (no staining), 1 (weak staining, light orange or green), 2 (moderate staining, medium orange or green), and 3 (strong staining, orange or green). Subsequently, the Sirius Red staining score was calculated through multiplying the percent positivity score and staining intensity score. Accordingly, the level of Sirius Red staining was defined as Low (0–4), Medium (5–8), and High (9–12).
Bioinformatics analysis of relapse-free survival of CRC patients
The CRC microarray profiles GSE39582 was used to analyze the correlation between the expression of COL1 or COL3 and the relapse-free survival of patients. The chip platform used in this analysis was the Affymetrix Human Genome U133 Plus 2.0 Array. The mRNA values of COL1 or COL3 were divided into low and high expression groups according to the median. Thus, each group had mRNA values for 283 cases. Then, the survival curves of the two groups were obtained using the Kaplan-Meier method.
Matrigel angiogenesis experiment
First, high concentration Matrigel (BD, 354248) was diluted in half. 10µl diluted Matrigel was pipetted into each well of µ-Slide Angiogenesis Glass Bottom (Ibidi, 81506) and was allowed to polymerize for 30 minutes at 37 ℃. 1X104 fibroblasts before or after CCL5 stimulation for 24h with 50ul medium were incubated on high concentration Matrigel. After 2h, the fibroblasts were fixed with 4% paraformaldehyde, stained with Hematoxylin and counted in 3 random visual fields using a light microscope. The capillary tubes were quantified by counting the number of lumens.
SPSS software for Mac OS version 25.0 was used for statistical analyses. An unpaired two-tailed Student’s t test was used to compare normally distributed data in two groups. Pearson’s χ2 test and Spearman’s correlation test were applied to analyze the correlation between the expression of CCL5 or Sirius Red staining and clinicopathological features. Log-rank test was performed in Kaplan–Meier survival curves. The correlated expression levels of CCL5 and Sirius Red staining in CRC tissue were analyzed by Spearman’s correlation test. All data were expressed as the mean ± standard error of mean (SEM). P < 0.05 was considered significant. ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001.