Animals
All experimental protocols were approved by the Institutional Committee for Animal Care and Use at the Technion, Israel Institute of Technology, Faculty of Medicine, Haifa, Israel. Approval number IL-157-10-21. All study procedures are compiled with the guidelines from the NIH Guide for the Care and Use of Laboratory Animals.
Age-matched C57BL/6 and NOD-SCID female and male mice (8–12 weeks old) were used. Mice were bred and raised at the Pre-Clinical Research Authority at the Ruth and Bruce Rappaport Faculty of Medicine. Surgery procedures were carried out under isoflurane anesthesia.
Tac Surgery
TAC surgery was performed on female and male mice (8–12 weeks old) as previously described 10. Aortic constriction was achieved using a 27-gauge blunt needle to create a standardized constriction of aorta. TAC-operation was performed by experienced veterinarian which was blinded to the experimental groups.
Echocardiography
Mice were anesthetized with 1% isoflurane and kept on 37⸰C heated plate during the procedure. Echocardiography was performed using the micro ultra sound Vevo3100 imaging system (VisualSonics, Fujifilm). Cardiac size, shape and function were analyzed by conventional 2-dimensional imaging and M-mode recordings. Maximal left ventricular end-diastolic (LVDd) and end-systolic (LVDs) dimensions were measured in short-axis M-mode images. Fractional shortening (FS) was calculated using the formula: FS (%)=[(LVDd-LVDs)/LVDd]*100. All values were based on the average of at least 3 measurements for each mouse.
Cancer Cells
The Polyoma Middle T (PyMT) murine breast carcinoma cell line was derived from the primary tumor-bearing transgenic mice expressing PyMT under the control of the murine mammary tumor virus promotor 11. The Lewis Lung Carcinoma (LLC) cells were purchased from the American Type Culture Collection (ATCC). Cells were tested and found to be free of mycoplasma contamination. Cells were cultured in DMEM supplemented with 10% Fetal Bovine Serum (FBS), 1% streptomycin and penicillin at 37⸰C in a humidified atmosphere containing 5% CO2.
PyMT and LLC cancer cells (105) were implanted subcutaneously into the flanks and ortho topically injected into the mammary fat pad or the back left side, respectively. Tumor volume was monitored over time and measured with a caliper. According to the Institutional Animal Care and Use Committee, the humane endpoint is defined when maximal tumor size reaches 1500 mm3.
Fibrosis Staining
Heart tissue was fixed in 4% formaldehyde for overnight, embedded in paraffin, serially sectioned at 10-µm intervals, and then mounted on slides. Masson trichrome staining was performed according to the standard protocol. Images were acquired using 3DHistech Panoramic 250 Flash III (3DHISTECH Ltd.). Each section was fully scanned. The percentage of the interstitial fibrosis was determined as the ratio of the fibrosis area to the total area of heart section using ImageJ software 12. Each dot represents the mean of the values taken from at least five fields, derived from a single mouse.
Immunofluorescence
Isolated hearts were fixed for 4 hours with 4% PFA at 4⸰C rinsed with PBS and incubated in 30% sucrose overnight before embedding in Optimal Cutting Temperature compound (OCT). Hearts were serially sectioned at 10µm intervals. Frozen heart sections were stained for F4/80 (Abcam 111101) and counterstained with DAPI, as previously described. Images were acquired with 3DHistech Panoramic 250 Flash III (3DHISTECH Ltd, Budapest, Hungary). Each section was fully scanned; for each analysis, 5 fields were randomly chosen and blindly analyzed with ImageJ software 12.
Rna Extraction And Quantitative Real-time Pcr
Total RNA was extracted from hearts using Aurum total RNA fatty or fibrous tissue kit (No. 732–6830, Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Next, cDNA was synthesized from 1000 ng purified mRNA with the iScript cDNA synthesis kit (NO. 170–8891, Bio-Rad). Real-time polymerase chain reaction was performed with QuantStudio3 (Thermofisher Scientific, 5823 Newton Drive, Carlsbad, CA, 92008, United States.). Serial dilutions were included for each gene to generate a standard curve. Values were normalized to the housekeeping gene Hsp90 expression levels. The primers sequences are listed in supplementary Table 1.
Hydroxyproline Assay
Hydroxyproline content was assessed as an index for collagen and fibrosis content in cardiac cell lysates by colorimetric hydroxyproline assay kit (Abcam, USA, Ab222941). The assay was performed according to the manufacturer's instructions.
Heart Single Cell Suspension And Flow Cytometry
Heart single cell suspension and flow cytometry was prepared as previously described 13. Briefly, hearts were perfused, extracted and finely minced. Then, incubated with digestive enzymes at 37oC on a rocking shaker at 50 rpm for 45–60 min. Samples were homogenized with 40µm cell strainer. Red blood cells were lysed using Ammonium-Chloride-Potassium (ACK) lysis buffer. Samples were centrifuged at 400xg for 5 min at 4 ⸰C and pellet was suspended with FACS buffer. Cells were immune-stained with the following anti-mouse antibodies: CD45- Alexa Fluor® 700 (BioLegend, 103128), CD11b- PerCP-Cy 5.5 (BioLegend, 101228), F480- PE (BioLegend, 123110) and CD206 – BV421 (BioLegend, 141717). cells were incubated (30 min, 4oC) with the antibody mixture in staining buffer (PBS containing 1% bovine serum albumin and 0.05% sodium azide), then washed twice with staining buffer. Cells were acquired using LSRFortessa flow cytometer (BD Biosciences). Data were analyzed using FlowJo V.10 software (FlowJo, Ashland, Oregon, USA).
Macrophage Depletion
Macrophage depletion was performed using the Macrophage depletion Kit using liposomes containing either Clodronate or saline as control (Encapsula Nanosciences LLS, USA, SKU# CLD-8901). Liposomes were injected IP according to the manufacturer's instructions. Clodronate treatment initiated together with cancer cell implantation followed by repeated injection twice a week until mice sacrifice at humane endpoint.
Statistics
Data are presented as mean ± SEM. All mice were included in each statistical analysis, unless they were euthanized for humane reasons before the experimental endpoint. Animals were chosen in randomized fashion for each experimental group. Statistical significance was determined by comparing between two means using Student t-test. Statistical analysis was performed with GraphPad Prism 8 software (La Jolla, CA). Values pf P < 0.05 were accepted as statistically significant.