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  • br Flank tumor transplantation studies br For

    2019-10-14


    Flank tumor transplantation studies
    For the ßank transplantation studies outlined below, investigators blinded themselves when possible to the assigned treatment group of each tumor for analysis; mice were de-identiÞed after completion of ßow cytometry analysis. The number of tumors transplanted for each study is based on past experience with studies of this nature, where a group size of 10 is sufÞcient to determine if pancreatic cancer growth is signiÞcantly affected when a regulatory signal is perturbed (see Fox et al., 2016).
    For shRNA-infected pancreatic tumor cell propagation in vivo, HG-9-91-01 were infected with lentiviral particles containing shRNAs and positively infected (red) cells were sorted 72 hours after transduction. 1000 low passage, shRNA-infected KPf/fC, or 2x105 shRNA-infected FG cells were resuspended in 50 mL culture media, then mixed 1:1 with matrigel (BD Biosciences). Cells were injected sub-cutaneously into the left or right ßank of 5-8 week-old NOD/SCID recipient mice. Subcutaneous tumor dimensions were measured with calipers 1-2x weekly for 6-8 weeks, and two independent transplant experiments were conducted for each shRNA at n = 4 in-dependent tumors per group.
    For drug-treated KPf/fC ßank tumors, 2x104 low passage REM2-KPf/fC tumor cells were resuspended in 50 mL culture media, then mixed 1:1 with matrigel (BD Biosciences). Cells were injected subcutaneously into the left or right ßank of 5-8 week-old non-tumor bearing, immunocompetent littermates or NSG mice. Tumor growth was monitored twice weekly; when tumors reached 0.1-0.3 cm3, mice were randomly enrolled in treatment groups and were treated for 3 weeks as described below. After 3 weeks of therapy, tumors were removed, weighed, dissociated, and analyzed by ßow cytometry. Tumor volume was calculated using the standard modiÞed ellipsoid formula ½ (Length x Width2); n = 2-4 tumors per treatment group in immunocompetent littermate recipients and n = 4-6 tu-mors per treatment group in NSG recipients.
    For chimeric transplantation studies, 2x104 low passage REM2-KPf/fC tumor cells were resuspended in 50 mL culture media, then mixed 1:1 with matrigel (BD Biosciences). Cells were injected subcutaneously into the left or right ßank of 5-8 week-old RORg-knockout or wild-type recipients; recipient mice were maintained on antibiotic water (sulfamethoxazole and trimethoprim). Tumor growth was monitored twice weekly; when tumors reached 0.1-0.3 cm3, mice were randomly enrolled in treatment groups and were treated for 3 weeks as described below. After 3 weeks of therapy, tumors were removed, weighed, dissociated, and analyzed by ßow cytometry. Tumor volume was calculated using the standard modiÞed ellipsoid formula ½ (Length x Width2); n = 5-7 tumors per treatment group.
    For drug-treated human pancreatic tumors 2x104 human pancreatic FG cancer cells or 2x106 patient-derived xenograft cells were resuspended in 50 mL culture media, then mixed 1:1 with matrigel (BD Biosciences). Cells were injected subcutaneously into the left or right ßank of 5-8 week-old NSG recipient mice. Mice were randomly enrolled in treatment groups and were treated for 3 weeks as described below. After 3 weeks of therapy, tumors were removed, weighed, and dissociated. Subcutaneous tumor dimensions were measured with calipers 1-2x weekly. Tumor volume was calculated using the standard modiÞed ellipsoid formula ½ (Length x Width2); at minimum n = 4 tumors per treatment group. In vivo and in vitro drug therapy
    at 20 mg/ml. For in vitro drug studies, low passage (< 6 passage) WT- or REM2-KPf/fC cells, (< 10 passage) KPR172H/+C cells, or FG cells were plated in non-adherent tumorsphere conditions or Matrigel colony conditions for 1 week in the presence of SR2211 or vehicle. For KPf/fC littermate, NSG mice, and RORg-knockout mice bearing KPf/fC-derived ßank tumors and for NSG mice bearing ßank patient-derived xenograft tumors, mice were treated with either vehicle (PBS) or gemcitabine (25 mg/kg i.p., 1x weekly) alone or in combination with vehicle (5% DMSO, 8% Tween80-PBS) or SR2211 (10 mg/kg i.p., daily) for 3 weeks. RORg-knockout mice and paired wild-type littermates were maintained on antibiotic water (sulfamethoxazole and trimethoprim). For NOD/SCID mice bearing ßank FG tumors, mice were treated with either vehicle (5% DMSO in corn oil) or SR2211 (10 mg/kg i.p., daily) for 2.5 weeks. All ßank tumors were measured 2x weekly and mice were sacriÞced if tumors were > 2cm3, in accordance with IACUC protocol. For KPf/fC autochthonous survival studies, 8 week old tumor-bearing KPf/fC mice were enrolled in either vehicle (10% DMSO, 0.9% NaCl with 0.2% acetic acid) or SR2211 (20 mg/kg i.p., daily) treatment groups, and treated until moribund, where n = 4 separate mice per treat-ment group. For all drug studies, tumor-bearing mice were randomly assigned into drug treatment groups; treatment group size was determined based on previous studies (Fox et al., 2016).