Animals
Female BALB/c Nude mice (C.Cg/AnNTac-Foxn1nu NE9) were purchased from Taconic Biosciences at 5 weeks of age and housed in an AALAC-accredited Small Animal Barrier Facility. Female B6 nude (B6NU) mice sp./sp. (B6.Cg/NTac-Foxn1nu NE10) were generated by breeding of male B6NU sp./sp. (B6.Cg/NTac-Foxn1nu NE10) with female B6NU sp./wt (B6.Cg/NTac-Foxn1nu NE10) mice. All animals and protocols were approved by the University of Pennsylvania IACUC review committee.
Study design
Female Balb/c Nude mice (5–8 weeks old) were inoculated in the 4th mammary fat pad with human breast cancer cells; while control mice received sterile phosphate buffered saline (PBS) and Matrigel. Mice that developed tumors received either PBS or combination cancer treatment (5 mg/kg doxorubicin (DOX), 4 mg/kg trastuzumab (TRZ), on alternate days by intraperitoneal injection) when tumors reached 100 mm3. Mice that did not receive xenografts got sham injections of sterile PBS. Mice were weighed twice per week and tumors were palpated/measured twice per week. Mice were maintained on chemotherapy/sham injections for the duration of the studies. A third study included mice that were injected with PBS and Matrigel only but were given DOX/TRZ treatment for 2 months for vessel permeability studies (Table 1 for all study designs). For Study I, cumulative doses of drug per mouse in cohort 1 was DOX = 40 mg/kg and TRZ = 32 mg/kg. In Study II, cumulative doses of drug for each mouse in cohort 1 was DOX = 80 mg/kg and TRZ = 64 mg/kg. In Study III, cumulative doses of drug for each mouse in cohort 1 was DOX = 40 mg/kg and TRZ = 32 mg/kg. In cohort 3, cumulative dose of drug for each mouse was DOX = 40 mg/kg.
Chemotherapy
Doxorubicin hydrochloride (DOX, Sigma-Aldrich D1515) was dissolved in sterile phosphate buffered saline (2 mg/ml), filter-sterilized, and diluted with sterile PBS to deliver 5 mg/kg in 100 μl, as an intra-peritoneal (IP) injection two times per week. Trastuzumab (TRZ, Herceptin™) was diluted with sterile PBS to deliver 4 mg/kg in 100 μl, as an IP injection once per week, on alternate days with DOX.
Xenograft procedure
BT474 cells (ATCC HTB-20), were plated onto 0.1% gelatin-coated T-175 culture flasks and grown to 70% confluence (exponential density). Cells were briefly harvested with 0.25% trypsin, washed with sterile PBS, counted, then injected in a volume of 200 μl as 2–5 X 106 cells mixed with an equal volume of phenol red-free Matrigel™ into the fourth left mammary fat pad. Controls received sterile PBS mixed with an equal volume of phenol red-free Matrigel.™ Mice were maintained under isofluorane anesthesia (2.5 ml/min) for 5 min/injection. Mice were weighed twice each week and received cell injections once they reached 18 g The fourth mammary fat pad is large enough to be felt through the skin of the BALB/c nude mouse, so injected cells with Matrigel™ were visible as a small bump until tumor growth became palpable. Tumors were measured with calipers twice a week and volumes were calculated using the formula V = (L X W2)/2, where L is tumor length and W is tumor width, in millimeters (mm) [19]. Chemotherapy was initiated when tumor volume surpassed 100 mm3 and was maintained throughout the study. Doxorubicin (5 mg/kg) and trastuzumab (4 mg/kg) were administered by intraperitoneal injection on successive days each week, while untreated mice received injections of sterile PBS.
Cell culture
Breast cancer cells
Her2+ human breast cancer cells (BT474) were purchased from the American Type Culture Collection (ATCC) and grown in culture according to the supplier’s directions. Human BT474 cells were grown in Hybri-Care Medium (ATCC) supplemented with 10% fetal bovine serum, L-glutamine and penicillin-streptomycin on 0.1% gelatin. Cells were passaged at 90% confluence and used in xenografts between passages 3–4.
Endothelial cells (EC)
Primary cultures of cardiac endothelial cells were prepared from hearts of 3–5 week-old C57BL/6 or B6NU mice using the protocol previously described for primary endothelial cell isolation from mouse testes [20]. Hearts were removed from euthanized mice and rinsed with sterile PBS to remove residual blood. Hearts were dissected free of great vessels, minced with scissors, and digested with 5 mg/ml type II collagenase (Worthington) for 30–45 min at 37 °C in a shaking incubator at 250 rpm. Collagenase digestion was quenched with an equal volume of FBS. Cells were filtered sequentially through 100 μm and 40 μm filters, washed 3 times with HBSS resuspended in DMEM/F12 supplemented with 10% FBS and pen-strep. Cells were pre-plated for 20 min to remove fibroblasts; non-adherent cells were washed with HBSS, resuspended in EC growth medium (Advanced DMEM supplemented with 15% FBS, EC growth supplement, L-glutamine, and pen-strep) and plated onto 0.1% gelatin-coated dishes overnight. The following day cardiac ECs were harvested by magnetic bead selection with anti-CD31 (Miltenyi Biotech) and expanded on 0.1% gelatin-coated dishes.
Endothelial cell assays
EC viability
Monolayer cultures of EC harvested from murine heart, lung, and liver were assessed for survival after 72 treatment of PBS, 0.1 μM or 1 μM doxorubicin using an MTS Viability Assay (Promega).
EC tube formation
Early passage [1, 2] heart or lung ECs were harvested with Accutase™ and plated onto basement membrane extract (BME, Cultrex™ R&D systems) at 5 μl/mm2 at a density of 1.8 X 103 cells/mm2 for EC tube formation. PBS or DOX (1 μM) was added to EC growth medium at 4 h and tube survival was documented by microscopy.
Echocardiography
Mice were anesthetized by inhalation 1.5–2% isofluorane via nose cone and placed on a heating pad to maintain body temperature; core temperature was monitored with a rectal probe. Echocardiography was performed using an MS400 probe on a Vevo2100 (FUJIFILM VisualSonics) by the Mouse Cardiovascular Phenotyping Core of the Penn Cardiovascular Institute, Perelman School of Medicine. Data were analyzed with the heart function package provided by VisualSonics Systems.
Histology
Hearts from study animals were flushed with PBS, fixed with 4% paraformaldehyde overnight, and maintained in 100% ethanol prior to dehydration and paraffin embedding (Molecular Cardiology Service Center, Penn Cardiovascular Institute, Perelman School of Medicine). Sections were stained for Masson’s Trichrome and Picrosirius Red (Direct Red 80, Sigma-Aldrich) using established methods. Images were acquired using an Olympus BX60 microscope equipped with Nikon DS-Fi2 and Qi-MC cameras and Nikon Elements processing software. Images of whole-heart histology were obtained with a Zeiss Axio Imager 2; images were motorized scanned and stitched with ZEN imaging software. Representative areas of each montage were used to calculate the percentage of fibrosis using ImageJ (NIH).
Miles permeability assay
Mice were injected intraperitoneally with 100 μl filter-sterilized Evans Blue (EB) dye (2% in sterile PBS) and allowed to move freely in the cage for 2 h, allowing dye circulation. Following euthanasia, hearts were flushed with PBS and cut in half along the anterior-posterior axis, to expose both ventricles and the interventricular septum. One half was flash-frozen in isopentane chilled in dry ice and the other half was weighed and placed in dimethylformamide to extract the EB. Samples were extracted for a minimum of 48 h at room temperature on a rotating shaker at 100 rpm. Samples were mixed with an equal volume of PBS and read as duplicates using black-walled 96-well plates, with an excitation wavelength of 620 nm and emission wavelength of 680 nm using a Biotek Synergy H1 plate reader. The amount of EB extravasated into the tissue was calculated from the measured fluorescence, relative to an EB standard curve, and expressed as μg EB/g tissue.
Fluorescence microscopy
Frozen sections of EB-labeled hearts were stained with rat anti-CD31 (BD Biosciences) primary antibody and goat anti-rat Alexa 488 (Invitrogen) secondary to visualize the endothelium of blood vessels. Extravasated EB dye was localized as red-orange fluorescence. Sections from unlabeled (no EB injection) samples served as controls for background fluorescence.
Enzyme-linked immunosorbent assay (ELISA)
Serum was prepared from whole blood samples taken from mice at the time of euthanasia prior to flushing hearts with PBS. The amount of cardiac troponin-I (cTnI) and brain natriuretic peptide (BNP) in serum was measured with ELISA kits from Life Diagnostics, Inc., and Ray Biotech, respectively, Samples were assayed in duplicate relative to purified mouse cTnI or BNP standard curves and read at 450 nm using a Synergy H1 plate reader (BioTek Instruments, Winooski, VT).
TUNEL assay
Apoptosis in frozen sections of heart tissue was detected using the Dead-End™ TUNEL System (Promega) to label fragmented DNA with fluorescein-12-dUTP according to the manufacturer’s protocol. Labeled sections and positive controls were observed by fluorescence microscopy (Olympus).
Protein analysis
Total protein was extracted from samples of hearts, lungs, and gastrocnemius muscles in RIPA buffer (Sigma-Aldrich, St. Louis, MO) and quantified using a Pierce BCA assay (ThermoFisher Scientific, Waltham, MA). Proteins were separated on 10% Bis-Tris NuPAGE gels in MOPS buffer (ThermoFisher) and transferred to Immobilon-P PVDF membranes (Millipore-Sigma). Blots were washed with Tris-buffered saline (TBS)-Tween 20 (10 mMTris, 0.9% NaCl, 0.1% Tween 20, TBS-T), blocked for non-specific binding with 5% nonfat milk in TBST, and incubated with primary antibodies to specific proteins, β-actin (Sigma-Aldrich A2668), ZO-1, occludin, claudin, and JAM-A (Santa Cruz Biotechnology, sc33725, sc133256, sc81796, and sc53624). Blots were washed in TBST and incubated with secondary antibodies (Cell Signaling Technology 7074, 98,164, and 91,196) linked to horseradish peroxidase (HRP). Protein bands were visualized using enhanced chemiluminescence (100 mM Tris pH 8.6, 0.2 mM p-coumaric acid, 1.25 mM luminol) documented with a FluorChemE imager (ProteinSimple, SanJose CA).
Statistical analysis
Sample means and standard errors were calculated and differences between experimental groups were compared using a Student’s t-test (GraphPad Prism 8.0). p < 0.05 was considered statistically significant.