Aminopropionitrile

For the generation of patient organoids, breast cancer surgical patients were consented under an IRB approved protocol for the USC-Palmetto Health Biorepository. TNBC organoids were established from a fresh surgical tissue by cutting the tumor into small pieces and incubating in collagenase A solution with ROCK inhibitor on a shaker at 37 °C for 30 min. The collagenase activity was inhibited by adding FBS, and pipetting was done to ensure the formation of almost a single cell solution. After several washes with PBS, the cell pellet was dissolved in matrigel. Breast organoid media containing ROCK and GSK inhibitors was added after the matrigel solidified^{69 (link)}. For the PDX organoid, a small, 2–3 mm³ piece of the PDX tumor that was freshly excised from an NSG mouse was minced and separated into single cell suspension as previously described^{70 (link)}. For drug testing studies, organoids were disrupted to single cells by digesting at 37 °C for 30 min with TrypLE (A1217701, Gibco, NY, USA) in the presence of 10uM Rock inhibitor (s1049, Selleckchem, TX, USA). Organoids were plated into wells of 96-well plate (20,000 cells/well) on a collagen-coated surface with media containing 2% matrigel (356252, Corning, NY, USA). Drugs were added 72 h after plating (final concentration of doxorubicin at 40 nM and BAPN at 25 mM). Cells were grown in the presence of drug(s) or vehicle for 9 days, and the diameter of organoids were measured.

To develop doxorubicin-resistant xenografts, 6–8-weeks-old female athymic nu/nu mice were injected with 2 × 10⁶ MDA-MB-231 cells into mammary fat pads (MFP). Mice were randomly allocated into two groups, and vehicle or doxorubicin treatments (5 mg/kg, weekly, i.v.) were started when tumors became palpable. Vehicle-treated tumors were collected and designated as vehicle. Half of the doxorubicin-treated mice were sacrificed when they were responsive to treatment, and tumors were collected and designated as sensitive. The remaining mice continued to receive doxorubicin until their tumors started regrowth. Then, these mice were also sacrificed, and tumors were collected and designated as resistant.
To test the role of LOX or FAK or Src inhibition to overcome chemotherapy resistance in immunocompetent setting, 2 × 10⁵ 4T1 cells were injected into the MFPs of 6–8 weeks-old Balb/c mice. Mice were treated with vehicle, doxorubicin (2.5 mg/kg, once a week, i.v.), BAPN (100 mg/kg, daily, ip.), PF-562271 (15 mg/kg, daily, oral), Saracatinib (25 mg/kg, daily, oral), or their combinations with doxorubicin. Once the vehicle group reached at 800 mm³, all mice were sacrificed, and tumor weight was measured.
For shLOX induction experiments using the luciferase overexpressing MDA-MB-231.Luc2GFP cells, doxycycline was given to induce shLOX expression at 100 ug/ml in drinking water when the tumors reach at 100 mm³. All mice were sacrificed; tumors were collected and weighed once the vehicle-treated tumors reached at 1500 mm³. For bioluminescence imaging, mice were intraperitoneally injected with 150 mg/kg D-luciferin (Perkin Elmer, MA, USA), and images were acquired with Lumina III In Vivo Imaging System (Perkin Elmer, MA, USA). Analysis was performed with Living image software by measuring photon flux.
For PDX experiments, 2–3 mm³ pieces of frozen PDX tumors were placed into the flank region of NSG mice. When tumors become palpable, mice were distributed into treatment groups. Doxorubicin (2 mg/kg, once a week, i.v.) and BAPN (100 mg/kg, daily, i.p.) was given individually or in combination for 30 days after which mice were sacrificed and tumors were collected for downstream analysis.
Primary tumor growth was monitored by measuring the tumor volume at least twice a week with a caliper after tumors became palpable. Tumor volumes were calculated as length × width²/2. All mice used were of the same age and similar body weight. All animal experiments have been approved by the Animal Ethics Committee of Bilkent University or the Institutional Animal Care and Use Committee of University of South Carolina. All mice were maintained under a temperature-controlled environment with a 12-h light/dark cycle and received a standard diet and water ad libitum.

8–12-week-old male C57BL/6 WT mice (Jackson Laboratory, Bar Harbor, ME) were used for this study. A topical elastase treatment murine model of AAA formation was used and phenotype was evaluated on day 14, as previously described (28 (link)). The abdominal aorta was treated topically with 30 μl of type 1 porcine pancreatic elastase (5 U/mg of protein) on day 0, with/without administration of Panx1 inhibitor, spironolactone (1.5, 5, or 50 mg/kg intraperitoneally) given from days 1 to 13. In a second model of chronic AAA which is associated with thrombus formation and aortic rupture, mice were treated with topical elastase and 0.2% β-aminopropionitrile (BAPN) with/without spironolactone treatment and analyzed on day 28. The change in aortic diameter was quantified by video micrometry on days 14 or 28 and expressed as percentage increase over baseline aortic diameter. Aortic phenotype were measured by video micrometry using NIS-Elements D5.10.01 software attached to the microscope (Nikon SMZ-25; Nikon Instruments, Melville, NY). Percentage increase in aortic diameter was determined by [(maximal AAA diameter–self-control aortic diameter)/(self-control aortic diameter)] × 100, and aortic dilation of ≥100% was considered positive for AAA formation.