Adherent Fluc-expressing cells grown in 15-cm tissue culture dishes were detached with trypsin, resuspended in DMEM with 10% fetal bovine serum (FBS), and centrifuged at 300g for 3 min. The cell pellets were then washed once with 1× PBS. The cells were resuspended in 1× PBS containing 2% FBS. Cell number and viability were assessed by automated cell counting on a Vi-CELL XR (Beckman Coulter). PBS (1×) containing 2% FBS was then added to achieve a cell concentration of 108/ml.

Female NCr nude mice at ~8 weeks old (Taconic, #NCRNU-F) were anesthetized by intraperitoneal injection of ketamine/xylazine. An incision was made in the skin, and 2 × 106 viable cells (20 μl) were injected through the fascia and into the lower pole of the renal parenchyma. The incision was closed using two to three wound clips. The mice were subcutaneously administered buprenorphine for analgesia immediately after wound closure and were allowed to regain movement and consciousness on a slide warmer. After surgery, the viability of the remaining uninjected cells was again assessed by counting on a Vi-CELL XR and was confirmed to be >98%. Mice were monitored daily for changes in weight, changes in activity, and food and water intake. Baytril was administered in drinking water for 7 days after surgery to prevent infection. Wound clips were removed 7 to 8 days after surgery.

Tumors were monitored weekly by BLI beginning 2 weeks after surgery (see below). Once the tumors showed at least two consecutive weeks of growth, the mice were randomized to receive abemaciclib (60 mg/kg), palbociclib (65 mg/kg), PT2399 (20 mg/kg), the combination of palbociclib (65 mg/kg) and PT2399 (20 mg/kg), or the corresponding vehicle(s), all by oral gavage daily for 28 days. The monotherapy mice also received the vehicle for the complementary drug used in the combination arm, and control mice received the vehicles for both combination partners. Imaging was performed by Animal Resources staff who were blinded to the treatment groups. Formulations were as follows: Abemaciclib was prepared in 1% hydroxyethyl cellulose/25 mM phosphate buffer, palbociclib was prepared in 50 mM sodium lactate buffer (pH 4.0), and PT2399 was dissolved in 10% ethanol/30% polyethylene glycol 400/60% water containing 0.5% methylcellulose and 0.5% Tween 80. Photon emission was normalized to the photon count on day 0 (the time of enrollment). Mice were sacrificed at the end of the dosing period for studies in which tumor weight was measured. For survival analysis studies, the mice were sacrificed when they lost 20% of their body weight or when they appeared moribund or distressed.

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