Carbonic anhydrase 9 ca9

All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Female 5- to 6-week-old Nu/Nu mice (Charles River) were housed in a sterile environment with micro isolator cages and allowed access to water and chow ad libitum. Mice were injected subcutaneously with 4×10⁶ WT or FBW7-KO HCT116 cells. After tumor growth for 7 days, mice were treated daily with regorafenib at 30 mg/kg by oral gavage for 10 consecutive days. Regorafenib was dissolved in Cremephor EL/95% ethanol (50:50) as a 4× stock solution. Tumor growth was monitored by calipers, and tumor volumes were calculated according to the formula 0.5 × length × width^{2 (link)}. Mice were euthanized when tumors reached 1.0 cm³ in size. Tumors were dissected and fixed in 10% formalin and embedded in paraffin. Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL; Millipore), active caspase 3 (Cell Signaling), CD31 (Spring Bioscience, Pleasanton, CA), and Carbonic Anhydrase 9 (CA9; Santa Cruz) immunostaining was performed on 5 μM paraffin-embedded tumor sections as previously described ^{33 (link)}, by using an AlexaFluor 488-conjugated secondary antibody (Invitrogen) for detection.