2.1 Reagents and facilities
RPMI-1640 was bought from Thermo Fisher Scientific corporation (Waltham, USA). Reagent extracting RNA was bought from Sangon Biotech (Shanghai, China). Cytometry labelled antibodies: PE anti-mouse F4/80 Antibody (Catalog: #123110, Biolegend, USA), FITC anti-mouse CD206 (MMR) Antibody (Catalog: #141704, Biolegend, USA), Fixable Viability Stain 450 (FVS450) (Catalog: #562247, BD Biosciences, USA). Recombinant mouse interleukin IL-4 protein (Catalog: #214 − 14, PeproTech, USA), Recombinant mouse Fstl1 protein (Catalog: #1738-FN-050, R&D systems, USA). Recombinant human TGFB1 protein (Catalog: #240-B-010/CF, R&D systems, USA), Recombinant human BMP4 protein (Catalog: #314-BP-010, R&D systems, USA).
2.2 Experimental animals
129 background Fstl1flox/+ mice were backcrossed to Balb/C background mice for more than 10 generations, and then our experimental animals were gained: Balb/C background Fstl1+/− mice. Experimental procedures of generation of Fstl1+/− mice were mentioned in previous report . For mouse genotyping, Fstl1 primer sequences were used, Forward: 5’-CTCCCACCTTCGCCTCTAAC-3’; Reverse: 5’-CGGCTAGGAAAGACTTGGAA-3’. Mice were purchased from the Model Animal Research Center of Nanjing University and were bred in our animal institution with controlled conditions, a 12h/12h light-dark cycle. All animal experiments were performed in accordance with the Administration Regulations on Laboratory Animals of Beijing Municipality. All animals were raised in specific pathogen free (SPF) level conditions and given adequate irradiated food and water. All used animals were healthy female Balb/C mice (6–8 weeks).
2.3 Experimental cell lines
4T1 breast cancer cell line is a kind gift from professor Wen Ning of Nankai University (Tianjin, China). Cells were grown in RPMI 1640 containing 10% fetal bovine serum (FBS) and 1% penicillin /streptomycin (P/S) and were cultured under 37°C and 5% CO2. Macrophage RAW264.7 cell line is a gift from professor Ying Sun, and Ana-1 cell line is a gift from professor De-Shan Zhou of Capital Medical University (Beijing, China). These two macrophage cell lines were cultured in sterile RPMI 1640 medium containing 10% fetal bovine serum (FBS) and incubated at 37°C, 5% CO2. Cells were cultured and generated for more than two generations and digested for following experiments.
2.4 Mouse genotyping
0.5-1cm mouse tail was cut and digested for genomic DNA solution. 50ul volume of DNA solution was removed from preceding solution, the double volume of 100% alcohol was added, and DNA was extracted visible to the naked eye. 500ml 75% alcohol was used to wash it, and finally, DNA was prepared to be diluted to a certain concentration.
2.5 Breast cancer model mice
6–8 week-old healthy female Balb/C background wild type mice (WT as control group) and Fstl1+/− mice (as experimental group) were age-matched and weight-matched to construct the animal model of breast cancer. On the 0th day, the mice were anaesthetized with pentobarbital sodium 0.25 mg/kg and injected 1×10^6 4T1 cells into the second fat pad under the stereoscope. The mice were subsequently housed and fed in individual ventilation cages (IVC). On the 28th day, mice were euthanized. Tumors in situ were cut off subsequently, tumor weights were measured and lungs were removed from mice thoracic cavity preparing for the following total mRNA, protein assay, and other experiments.
2.6 Quantitative real-time polymerase chain reaction (q-PCR) and reverse transcription polymerase chain reaction (RT-PCR)
β-actin primers, Forward: 5’-CATCCGTAAAGACCTCTATGCCAAC-3´; Reverse: 5’-ATGGAGCCACCGATCCACA-3´; IL-4 primers, Forward: 5´-GGTCTCAACCCCCAGCTAGT-3´; Reverse: 5´-GCCGATGATCTCTCTCAAGTGAT-3´; IL-13 primers, Forward: 5´-CCTGGCTCTTGCTTGCCTT-3´; Reverse: 5´-GGTCTTGTGTGATGTTGCTCA-3´; Arg-1 primers, Forward: 5´-CAAGGTGATGGAAGAGACCTT-3´; Reverse: 5´-TAAGGTAGTCAGTCCCTGGCTT-3´; IL-10 primers, Forward: 5´-ATGCTGCCTGCTCTTACTGACTG-3´; Reverse: 5´-CCCAAGTAACCCTTAAAGTCCTGC-3´; CCL2 primers, Forward: 5´- TTAAAAACCTGGATCGGAACCAA-3´; Reverse: 5´- GCATTAGCTTCAGATTTACGGGT-3´; CCL5 primers, Forward: 5´-GCTGCTTTGCCTACCTCTCC-3´; Reverse: 5´-TCGAGTGACAAACACGACTGC-3´;
CSF-1 primers, Forward: 5´-GTGTCAGAACACTGTAGCCAC-3´; Reverse: 5´-TCAAAGGCAATCTGGCATGAAG-3´; VEGF-α primers, Forward: 5´- GCACATAGAGAGAATGAGCTTCC-3´; Reverse: 5´-CTCCGCTCTGAACAAGGCT-3´; TGF-β primers, Forward: 5´- CTCCCGTGGCTTCTAGTGC-3´; Reverse: 5´-GCCTTAGTTTGGACAGGATCTG-3´. PCR analyses was performed as described previously .
2.7 Western blot analyses
The lung tissues and breast tissues were lysed in RIPA buffer (Applygen, Beijing, China) containing a proportional mixture of protease inhibitor (1:50) and protein phosphatase inhibitor (1:100). Detection of mouse Fstl1 antibody (Catalog: AF1738, R&D Systems, dilution 1:1000), β-actin (Catalog: 4970, Cell Signaling Technology, dilution 1:2000), Arg-1 (Catalog: sc-271430, Santa Cruz Biotechnology, dilution 1:1000), TGF-β (Catalog: ab66043, Abcam, dilution 1:1000), Smad2/3 (Catalog: 8685, CST, dilution 1:1000), P-Smad2/3 (Catalog: 8828S, CST, dilution 1:1000), Smad1/5/8 (Catalog: sc-6031-R, Santa Cruz, dilution 1:1000), P-Smad1/5/8 (Catalog: sc-12353, Santa Cru, dilution 1:1000), Smad1/5 (Catalog: sc-6031-R, Santa Cruz, dilution 1:1000); P-Smad1/5 (Catalog: 9516T, CST, dilution 1:1000), MMP-9 (Catalog: ab58803, Abcam, dilution 1:1000) was performed. Western blot analyses were performed using standard protocols as described previously .
2.8 Flow cytometry
Cytometric analyzed single-cell suspensions were prepared from lung tissues. The lung tissues were cut into 2–4 mm3 and placed in a sieve for mechanical digestion. To identify immune cell populations, cells were first incubated with Fixable Viability Stain 450 (FVS450) for 15 minutes, and then they were incubated with a monoclonal antibody CD16/32 for 10 minutes to block cellular Fc receptor. Ultimately cells were stained with PE anti-mouse F4/80 antibody and FITC anti-mouse CD206(MMR) antibody for 60 minutes. CD206 is a transmembrane protein and needed both cellular surface staining and intracellular staining. If necessary, the samples were sustained overnight using 2% paraformaldehyde (PFA). Data were integrated by Tree Star Flowjo software.
2.9 Macrophage migration experiment
To mimic the lung tumor microenvironment, the transwell (pore size: 8.0µm) was used to study the co-culture of 4T1 breast cancer cells and RAW264.7/Ana-1 macrophages. RAW264.7/Ana-1 cells (3×10^4 cells/well, 150ul 1640) were placed in the upper well while 4T1 cells (1.2×10^5 cells/well, 500ul complete medium containing 10%FBS) were placed in the lower well. RAW264.7/Ana-1 macrophages:4T1 cells = 1:4. Fstl1 and IL4 were administered into the lower solution separately or both. After 12h co-culture, the 8.0µm transwell was washed and stained by 4% paraformaldehyde (PFA) containing 0.1% crystal violet for 30 mins. 5 fields per slide were observed with 100× magnification and migrated macrophages were calculated using Photoshop software compared with blank control.
2.10 Cell proliferation assays
4T1 breast cancer cells were grown and digested by 0.25% trypsin for cell proliferation analysis. 4T1 cells (1.2×10^5 cells/well) were inoculated in 96-well plates and were cultured at 37°C, 5% CO2 for three time points (12h, 24h, 48h), treated with different concentrations of recombinant mouse Fstl1 protein (0ng/ml, 250ng/ml, 500ng/ml and 1000ng/ml). Ultimately, the absorption data were assayed by enzyme-labelled instrument for every single time point under the particular OD value (450nm).
2.11 Hematoxylin-Eosin staining
Paraffin embedded lung tissue was cut into sections. The lung tissue sections were put into distilled water and were dyed in hematoxylin solution for several minutes. Then they were put into the acid water and ammonia water for color separation, a few seconds each. Followed rinsing with water for 1 hour and then with distilled water for a while. Sections were dehydrated in 70% and 90% alcohol for 10 minutes each and stained by alcohol eosin staining solution for 2–3 minutes. The stained sections were dehydrated by pure alcohol and were transparented by xylene. Finally, dropping gum on the sections and putting the cover glass to seal it.
2.12 Wound healing experiment
4T1 cell migration was evaluated by the wound healing experiment, also known as the “scratch” assay. The experiment was divided into 3 groups including 4T1 group, 4T1 negative control (4T1NC) group and 4T1 overexpressed (4T1OE) group. When the cells reached 90% confluence, a scratch was made through each well using a 100ul sterile pipette tip. Cells were monitored with 100× magnification at 0h, 12h and 24h after wounding. Images of cells were captured at the same marked position to document the repair process. The experiment had been repeated for 3 times.
2.13 Immunohistochemistry (IHC) assay
Firstly, lung samples were pretreated in 4% formaldehyde fixed for 24h and were immersed in 70% alcohol saved for 24h. Paraffin-embedded lung sections were deparaffinized in xylene, dehydrated through gradient ethanol and then heated in citrate buffer (pH ≈ 6.0) for antigen retrieval. After blocked with 3% H2O2 for 15 mins, the tissue sections were incubated at 4°C overnight with Arg-1 antibody (Catalog: sc-271430, Santa Cruz Biotechnology, dilution 1:500), followed by incubation with HRP-conjugated secondary antibody (ZSGB-bio, Beijing, China) at 37°C for 30 mins. After washing three times with PBS, color was developed using DAB Chromogen (ZSGB-bio, Beijing, China). Slides were rinsed in tap water and counterstained with hematoxylin. Five random fields per section were viewed under the microscope.
UALCAN is used for digging TCGA (The Cancer Genome Atlas) data and CPTAC (Clinical Proteomic Tumor Analysis Consortium) data. Website address: http://ualcan.path.uab.edu. Kaplan-Meier survival analysis is capable to access the survival rate of 21 cancer types including breast cancer. Website address: https://kmplot.com/analysis/.
2.15 Statistical analyses
Statistical analysis was performed using Microsoft excel 2019 (Microsoft Corp.), GraphPad Prism 6.0 (GraphPad Software Inc.) and Image J. All results were calculated as mean ± standard deviation and all analysis were considered p < 0.05 statistically significant. All graphs show mean ± SD.