Tissue microarray construction and CAF assessment by immunohistochemistry (IHC)
IHC was performed by using human breast cancer microarrays of formalin-fixed paraffin-embedded (FFPE) tissues (Alianna, Xi an, China), and isolated fibroblasts were stained with antibodies against human α-smooth muscle actin (α-SMA) (ab5694; Abcam, Cambridge, UK) and FAP (ab28244; Abcam). Antibodies (1:100 dilutions) were incubated at 4 °C overnight. Antibody staining was developed using the Vectastain ABC kit (#PK-4000) and DAB (#SK-4100) detection system (Vector Laboratories, CA) and accompanied by hematoxylin counterstaining. Scoring for each immunohistochemistry marker was performed by two experienced technologists who were blinded to the results of other markers or case identity.
Isolation of primary fibroblasts
CAFs were isolated from human invasive mammary ductal carcinoma tissues, and paracancer fibroblasts (PCFs) were from a region at least 3 cm away from the outer tumor margin in the same patient as the CAFs. Fibroblasts from fibroadenoma (FADs) and non-cancer-associated fibroblasts (NAFs) were isolated from a reduction mammoplasty, in which only normal mammary tissue was detectable. All tissues were minced with scalpels and then enzymatically dissociated in mammary epithelial basal medium (Lonza, USA) supplemented with 2% bovine serum albumin (Promega, USA), 10 ng/mL cholera toxin (Sigma-Aldrich is now Merck KGaA, Darmstadt, Germany), 300 units/mL collagenase (Invitrogen, Carlsbad, CA, USA), and 100 units/mL hyaluronidase (Sigma-Aldrich is now Merck KGaA, Darmstadt, Germany) at 37 °C for 18 h. On the second day, the trypsinized suspension was centrifuged at 700 rpm for 5 min to separate the epithelial and fibroblast cells. The supernatant was collected for centrifugation at 800 rpm for 10 min to pellet the fibroblasts, followed by two washes with DMEM/F12 medium. The cell pellet was resuspended in DMEM/F12 medium supplemented with 5% FBS (GIBCO, Carlsbad, CA, USA) and 5 μg/mL insulin (Tocris Bioscience), plated in cell culture flasks and maintained undisturbed for 2 to 5 days. All tissues were obtained from the Ruijin Hospital with approval of the hospital ethical committee and by the patients’ written informed consent (Shanghai, China).
Collection of conditioned media (CM) and chemiarray
The CM of all types of fibroblasts was obtained after 48 h of conducting parallel cell culture experiments. The CM samples were then centrifuged at 4000 rpm for 10 min to remove the insoluble substances. Two milliliters of CM were then used for the chemiarray protocol, which is described in the Human Cytokine Antibody Array Kit (RayBiotech, Norcross, GA, USA).
Enzyme-linked immunosorbent assay (ELISA)
Quantification of IL-6 levels in the supernatants of fibroblasts or breast cancer cells was carried out by ELISA according to the protocol of the human IL-6 Sandwich immunoassay kit (capture IL-6 antibody #MAB206, detection IL-6 antibody #BAF206 and standard rhIL-6 #206-IL; R&D Systems, Minneapolis, MN, USA). All samples were quantified in multiple wells per experiment and repeated three times.
Cell culture
The human BrCA cell lines MCF7, SK-BR-3, BT-474 and MDA-MB-231 were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (HyClone, Waltham, MA, USA) or RPMI-1640 (HyClone) supplemented with 10% FBS (GIBCO, Carlsbad, CA, USA) and 1% penicillin/streptomycin (GIBCO). Cells were cultured at 37 °C in an incubator with a 5% CO2 atmosphere. Cells were treated with recombinant human IL-6 (#HZ-1019, HumanZyme, Chicago, USA) and STAT3 inhibitor (#S3I-201, Selleckchem, USA) at the indicated concentrations in each manipulation.
Western blot
Cells were washed 3 times with PBS and treated with RIPA lysis buffer (#89900, Thermo Fisher, Waltham, MA, USA) mixed with protease and phosphatase inhibitor (Roche, Basel, Switzerland). Ten to twenty micrograms of total protein from each sample was resolved on a 10% PAGE gel and transferred to a polyvinylidene difluoride (PVDF, Merck Millipore, Germany) membrane. The blots were then probed with antibodies against GAPDH (1:10000, KangChen, Shanghai, China), STAT3 (1:1000, #4904, Cell Signaling Technology, USA), pSTAT3 (Tyr705) (1:1000, #4903, Cell Signaling Technology, USA), HIC1 (1:5000, #H8539, Sigma-Aldrich, Saint Louis, MO, USA) and cyclin D1 (1:1000, #2978, Cell Signaling Technology), followed by incubation with peroxidase-labeled secondary antibodies. Immunoreactive proteins were detected by enhanced chemiluminescence (ECL) detection kit (Merck Millipore, Germany).
Cell counting Kit-8 (CCK8) for the cell proliferation assay
Proliferation assays of MCF-7, BT-474, SK-BR-3 and MDA-MB-231 cells treated with different media (supernatant of NAF and CAF) were performed with CCK8 (Dojindo, Rockville, MD). Briefly, cells were cultured in 96-well plastic plate wells in different media for 2 and 4 days, followed by labeling with CCK8 (1:10 dilution) for one additional hour. The absorbance of the samples was measured on a VersaMax Microplate Reader at a wavelength of 450 nm. All experiments were carried out with five parallel wells and repeated 3 times.
Flow cytometry
BrCA cells were trypsinized and resuspended in PBS containing 2% heat-inactivated FBS and blocked for 10 min with FcR reagent. Then, APC-labeled anti-IL-6Rα antibody (anti-human CD126, #561696, BD Pharmingen, USA) was added and incubated for 30 min on ice in the dark. Thereafter, cells were washed twice with PBS and then analyzed on a FACSCalibur Flow Cytometer (Becton Dickinson, San Jose, USA).
Cell cycle analysis
Cells in 6-well plates cultured with NAF and CAF were trypsinized, washed and fixed in 70% ethanol for 48 h at 4 °C. The nuclei were stained with propidium iodide (PI, 50 μg/ml) in 1% Triton X-100/PBS containing 100 μg/ml DNase-free RNase, and the DNA content was measured by flow cytometry with the FACSCalibur platform (Becton Dickinson, San Jose, USA). The proportion of cells in the different cell cycle phases was calculated using the ModFit LT program (Verity Software House, USA).
Colony formation assay
In this assay, one hundred SK-BR-3 cells were plated into each well of a 12-well plate and cultured for 21 days, with an additional equal volume of NAF or CAF supernatant. At the end of the culture period, supernatants were removed and cells were fixed with methanol for 30 min and stained with crystal violet for 30 min. Next, the plates were washed several times with water gently, and images of the optical density of the cells were captured by a digital camera. The stained cell area was measured by Image-Pro Plus 6.0 to determine the cell proliferation level. The MDA-MB-231shIL-6 test was performed with a similar method.
Real-time PCR
Total RNA was extracted from the cells using TRIzol reagent (#15596–026, Invitrogen) and reverse transcribed using the PrimeScript 1st Strand cDNA synthesis kit (#6110A, TaKaRa, China). Real-time PCR was conducted by using the FastStart Universal SYBR Green Master (Rox) (#04913850001, Roche) and Applied Biosystems 7500 Fast Real-Time PCR System (ABI, USA). All results were normalized to the GAPDH internal control. The sequences of the primers that we used were as follows: GAPDH-F: GGAGCGAGATCCCTCCAAAAT, GAPDH-R: GGCTGTTGTCATACTTCTCATGG, IL-6-F: ACTCACCTCTTCAGAACGAATTG, IL-6-R: CCATCTTTGGAAGGTTCAGGTTG.
IL-6 knockdown and lentivirus packaging
IL-6 knockdown was achieved by constitutively expressing shRNA targeting IL-6 in MDA-MB-231 cells using lentivirus. pLVX-shRNA2 lentiviral vectors expressing the fluorescent protein ZsGreen1 were used (Clontech, Mountain View, CA, USA), and the shRNA sequences were as follows: si-IL-6-1, 5′-CTCAAATAAATGGCTAACTTA-3′. Lentivirus packaging and cell sorting of transfected cells were routinely followed as previously described [24].