Antibodies, small interfering RNA (siRNA), and primer sequences
The primary antibodies used in the study are summarized in Additional file 6: Table S1. The siRNA and primer sequences used in the study are summarized in Additional file 7: Table S2 and Additional file 8: Table S3 respectively.
Cell lines and cell culture
The human 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), and HCT-15 (CCL-225); the human normal colorectal epithelial cell line FHC (CRL-1831); and the human normal colorectal fibroblast cell line CCD-18Co (CRL-1459) were all purchased from the American Type Culture Collection (ATCC). All human CRC cells were cultured in RPMI-1640 medium (Gibco, C11875500BT) supplemented with 10% fetal bovine serum (FBS) (ExCell Bio, FND500). FHC was cultured in RPMI-1640 medium supplemented with 15% FBS, and CCD-18Co was cultured in Eagle’s Minimum Essential Medium (EMEM) (ATCC, 30–2003) supplemented with 10% FBS. All cells were cultured at 37 °C in humidified atmosphere containing 5% CO2. Cell line certificates of analysis were obtained from the ATCC. All cell lines were negative for mycoplasma.
Primary normal colorectal fibroblasts: extraction and culture
Fresh human normal colorectal mucosae were cut using surgical scissors and then enzymatically dissociated in a mixture of type IV collagenase (2.0 mg/ml, Sigma, C5138), hyaluronidase (0.4 mg/ml, Sigma, H1115000) and DNase (25 U/ml, Solarbio, D8071) at a constant temperature of 37 °C for 2 h. The tissues were then passed through a 40-μm cell strainer to generate a single-cell suspension. The cell suspensions were centrifuged at 300 × g for 12 min; the supernatant was discarded, and the cell pellet was resuspended in EMEM. Primary cells were then plated at a density of 1 × 105 viable cells in 25 cm2 adherent flasks and cultured at 37 °C in EMEM with 10% FBS in humidified atmosphere containing 5% CO2 (enzyme digestion method). The tissues that could not pass through the strainer were transferred to 25 cm2 adherent flasks. EMEM supplemented with 10% FBS was added to the adherent flasks after 24 h, when the tissues had stuck to the bottom. The tissues were incubated until fibroblasts crawled out of them (improved tissue planting method).
Clinical CRC 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.
Identification and quantification of tumor budding
Tumor budding was identified based on the presence of a single tumor cell or a tumor cell cluster of up to 4 cells at the invasive front of CRC tumors. The quantification of tumor budding was performed according to five procedures proposed by the International Tumor Budding Consensus Conference (ITBCC) 2016 for reporting tumor budding in CRC during daily diagnostic practice .
IHC was performed using paraffin-embedded sections of human CRC tissue following the standard LSAB protocol (Dako). Primary antibodies against α-SMA, CD90, FAP, CCL5, SLC25A24, CD31, and VEGFA were used for IHC. The degree of staining was observed and scored independently by three pathologists. The percent positivity of CCL5 staining was scored from 0 to 4, as follows: 0 (< 5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (> 75%). The staining intensity was scored on a 4-point scale, as follows: 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 by multiplying the percent positivity score with the staining intensity score. Accordingly, the expression of CCL5 was classified as low (0–4), medium (5–8), or high (9–12).
Immunofluorescence (IF) analysis of cells and CRC tissue
IF analysis of cells was performed as previously described . For IF analysis of CRC tissue, the steps before primary antibody incubation were the same as those used for IHC. The steps after primary antibody incubation were the same as those used for IF analysis of cells. Images of cells were acquired using a laser scanning confocal microscope (Olympus, Japan), and images of CRC tissue were acquired using a fluorescence microscope (Olympus, Japan).
Boyden transwell chambers (Corning, 353,097) were used according to manufacturer’s instructions. Briefly, 2 × 104 fibroblasts were added to the upper chambers. The lower chamber contained of the following: 1 × 105 human CRC tumor cells, different concentrations of human CCL5 (Peprotech, 300–06-20), or the conditioned medium (CM) samples from stable CRC cell lines. After 48 h of incubation, fibroblasts that successfully migrated to the lower chamber were fixed with 4% paraformaldehyde and stained with hematoxylin. The number of cells was counted in five random visual fields using a light microscope (Olympus, Japan).
Human cytokine array
Serum-free CM samples from FHC and the human CRC tumor cell lines HCT-8, HCT116, HCT-15, and SW620 were collected after incubation for 24 h and filtered through a 0.22-μm mesh. The CM samples were added to arrays containing antibodies against 1000 unique cytokines (RayBio, GSH-CAA-X00) and processed according to manufacturer’s instructions.
RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total cellular mRNA was extracted using the TRIzol reagent (TaKaRa, 9109). The Prime-Script RT Reagent Kit with gDNA Eraser (TaKaRa, D6110A) was used to reverse-transcribe mRNA into cDNA. Finally, the SYBR Premix Ex Taq (TaKaRa, RR420) and the Applied Biosystems™ 7500 Fast Real-Time PCR System (Thermo Fisher, USA) were used for qRT-PCR. All mRNA levels were normalized based on GAPDH levels, and the 2−ΔΔCt method was used.
Enzyme-linked immunosorbent assay (ELISA)
CCL5 supernatant levels in the CM samples from FHC and human CRC tumor cells were measured with ELISA using a commercially available kit (CUSABIO, CSB-E17375h), as described by the manufacturer’s instructions. The results were expressed in pg/ml, and the standard curve was based on the measured OD values of the standard.
siRNAs for human CCL5, CCR1, CCR3, CCR4, CCR5, CD44, GPR75, and SLC25A24 were purchased from GenePharma (Suzhou, China). HCT-8, SW620, CCD-18Co, and human primary normal colorectal fibroblasts were transiently transfected with siRNA using the Lipofectamine 3000 Transfection Reagent (Invitrogen, L3000-015) based on the manufacturer’s instructions.
Construction of stable cell lines
The lentivirus vector LV17 (EF-1a/Luciferase17&Puro) carrying the human CCL5-overexpressing sequence (CCL5) and the lentivirus vector LV16 (U6/Firefly&Puro) carrying the indicated CCL5-repressing short hairpin RNA (shRNA) sequence (GGGTTCGGGAGTACATCAA) (shCCL5) were obtained from GenePharma (Suzhou, China). Empty LV17 and LV16 vectors served as controls for the overexpression (Vec) and repression vectors (shCtrl), respectively. Based on the manufacturer’s instructions, stable cell lines were established by transfecting human CRC cell lines with these lentiviral vectors.
Orthotopic CRC xenograft mouse model
BALB/C-nude mice (male, 3–5 weeks old) were purchased from GemPharmatech Co., Ltd (Guangdong, China). They were housed under specific pathogen-free conditions in the animal facility at the Shenzhen Hospital, Southern Medical University, China. All animal experiments were approved by the ethics committee of Shenzhen Hospital, Southern Medical University, China. First, 5 × 106 HCT116, SW620/shCtrl, or SW620/shCCL5 cells were subcutaneously injected into the backs of nude mice (n = 3). After 2 weeks, the IVIS Spectrum In Vivo Imaging System (PerkinElmer, USA) was used to image tumor progression in mice with SW620/shCtrl and SW620/shCCL5 xenografts. Further, 15 mg/ml D-Luciferin potassium salts (Promega, P1043) were intraperitoneally administered to the mice (dose, 10 μl/g). The mice were sacrificed, and tumors were surgically removed, fixed in 10% formalin, embedded in paraffin, and cut into 2.5-μm-thick sections for hematoxylin–eosin (H&E) staining.
After the subcutaneous tumors of HCT116 formed, one part of tumor tissues was fixed and stained with H&E, and the remaining tumor tissues were removed and cut into 1-mm3 pieces using ophthalmic scissors. Of these pieces, five were chosen and buried inside the cecal serosal layer in nude mice using purse string sutures (n = 5). After 8 weeks, the ceca of nude mice were surgically removed and processed as mentioned above.
Immunoblot/western blot (WB)
Total proteins were isolated from cells using the 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). The total proteins were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk (FDbio, FD0080) or 5% BSA (FDbio, FD0030) for 1 h at 25 °C and then incubated with primary antibodies at 4 °C overnight. Subsequently, the membranes were incubated with a goat anti-rabbit or anti-mouse secondary antibody (FDbio, FDR007 and FDM007). The proteins were detected using an ECL chemiluminescence solution (FDbio, FD8030) and visualized using a chemiluminescence detection system (Universal Hood II, Bio-Rad). The intensity of each immunoblot band was quantified using the NIH Image J software (National Institutes of Health, USA).
Treatment with CCR1, CCR5, and Akt inhibitors
Before the recruitment assay, fibroblasts were pre-cultured with a CCR1 inhibitor (BX471, 100 nM, MedChemExpress, HY-12080A), CCR5 inhibitor (Maraviroc, 100 nM, Selleck, S2003), or Akt inhibitor (MK-2206, 2 μM, Selleck, S1078) for 48 h. During the recruitment assay, BX471, Maraviroc, or MK-2206 was added to both the upper and lower chambers.
Total mRNA was extracted from fibroblasts before and after treatment with 40 ng/ml CCL5 for 24 h. Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations, and index codes were added to attribute the sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions.
Matrigel angiogenesis experiment
First, the high-concentration Matrigel (BD, 354,248) was diluted to half the original concentration. Then, 10 μl of this diluted Matrigel was added to each well of a µ-Slide Angiogenesis Glass Bottom (Ibidi, 81,506) and allowed to polymerize for 30 min at 37 °C. Subsequently, 50 μl medium containing 1 × 104 fibroblasts (treated or not treated with 40 ng/ml CCL5 for 24 h) was incubated in the diluted high-concentration Matrigel. After 2 h, the fibroblasts were fixed with 4% paraformaldehyde and stained with hematoxylin. The number of lumens was obtained across three random visual fields using a light microscope, and capillary tubes were quantified by counting the number of lumens.
Flow cytometry (Flow-Cyt)
A 4 °C centrifuge was pre-cooled, and PBS containing 0.1% BSA was prepared in advance. Fibroblasts treated with 40 ng/ml CCL5 for 24 h and untreated fibroblasts were collected and washed with PBS. These cells were incubated with the APC-VE-cadherin antibody or APC-rabbit-IgG antibody at 4 °C for 30 min under dark conditions. Then, the cells were washed again with PBS. Finally, the cells were resuspended in 100 μl of PBS, and the cell suspension was transferred to a flow cytometry tube for detection. The fluorescence intensity of fluorescein isothiocyanate was quantified using the Flow-jo software (BD, USA).
Sirius red staining
Sirius Red staining was performed on paraffin-embedded sections of human CRC tissue and tumor tissue from mouse caecum using the Sirius Red Staining Kit (LEAGENE, DC0041) based on the manufacturer’s protocol. The degree of staining was observed under a polarized light microscope (Olympus, Japan) and scored independently by three pathologists. The percent positivity of Sirius Red staining was scored from 0 to 4, as follows: 0 (< 5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (> 75%). The staining intensity was scored on a 4-point scale, as follows: 0 (no staining), 1 (weak staining, light orange and/or green), 2 (moderate staining, medium orange and/or green), and 3 (strong staining, orange and/or green). Subsequently, the Sirius Red staining score was calculated by multiplying the percent positivity score with the staining intensity score. Accordingly, the level of Sirius Red staining was categorized as low (0–4), medium (5–8), or high (9–12).
Bioinformatics analysis of relapse-free survival and correlation with mRNA expression in CRC patients
The human CRC microarray profile GSE39582 was used to analyze the correlation of COL1 and COL3 expression with relapse-free survival in CRC patients. The chip platform used in this analysis was the Affymetrix Human Genome U133 Plus 2.0 Array. Based on the median values of COL1 and COL3 mRNA expression, patients were divided into low and high expression groups. Each group contained mRNA values for 283 patients. Then, the survival curves of the two groups were obtained using the Kaplan–Meier method. In addition, the human CRC microarray profile GSE39582 was also used to analyze the correlation of CCL5 expression with COL1 and COL3 expression.
Scanning electron microscopy
Fresh subcutaneous tumor tissues from nude mice (≤ 3 mm3) were surgically removed immediately after the mice were sacrificed. The tissues were promptly added to a fixative solution for electron microscopy (Servicebio, G1102) and incubated for 2 h at room temperature before being transferred to 4 °C for storage. Subsequently, the samples were observed and photographed using a scanning electron microscope (Hitachi, Japan).
SPSS software for Mac OS version 25.0 (IBM, USA) was used for statistical analyses. An unpaired two-tailed Student’s t test was used to analyze the differences between two groups. The Mann–Whitney U test was conducted to compare the scores of CCL5 staining or Sirius Red staining between CRC tumor tissues and adjacent normal tissues. Pearson’s χ2 test and Spearman’s correlation test were applied to analyze the correlation of CCL5 expression or the level of Sirius Red staining with clinicopathological features. The log-rank test was performed to analyze Kaplan–Meier survival curves. The correlation of CCL5 expression with the level of Sirius Red staining in CRC tissues, and the correlation of CCL5 mRNA expression with COL1 or COL3 mRNA expression in the human CRC microarray profile GSE39582 were analyzed using Spearman’s correlation test. All data were expressed as the mean ± standard deviation (SD). P < 0.05 was considered statistically significant (ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001).