Cells are plated overnight and treated with 100 nM AZD3965 or vehicle for 24 hours. The cells are then washed, once in PBS and twice with lysis buffer (50 mM Mops, 100 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 0.1 mM DTT, 1 mM PMSF supplemented with 1× mini complete protease inhibitor cocktail tablets. The cells are harvested by scraping and centrifugation, and the pellet snap frozen without buffer in liquid nitrogen. Frozen aliquots of cells are thawed on ice and re-suspended in lysis buffer. Cells are lysed by 3 rounds of freezing in liquid nitrogen and thawing at 37°C for 2 minutes each. Lysates are subsequently centrifuged (13000 g, 10min, 4°C) until clear and then stored on ice. Enzyme activity in the cell lysates is determined using a micro-plate reader to measure either production or depletion of NADH/NADPH, through its absorbance at 340/10 nm, occurring as a result of direct or coupled enzyme reactions. The 96 well plates used for the assays are pre-heated to 37°C for 10 minutes prior to starting reactions, initiated by the addition of 5 μL cell lysate to 95 μL of reaction buffer (50 mM Mops pH 7.4, 100 mM KCl, 5 mM free magnesium). The standard reaction buffer is supplemented to assay the kinetics of the different enzymes. Absorbance values for controls are subtracted from absorbance of corresponding reactions. Graphpad prism 6 is used to plot V₀ values against substrate concentration and determine V_max and K_m values. The V_max is then normalised to the protein concentration in the cell lysate^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[2]
COR-L103 xenografts are grown by subcutaneous injection of 5×10⁶ cells in 0.2 mL of 1:1 serum-free RPMI:Matrigel into the mid-dorsal flank of 8 to 14-week-old male NOD scid gamma mice. Mice are housed in individually vented caging systems in a 12-hour light/12-hour dark environment and maintained at uniform temperature and humidity. Tumor size is measured twice a week using calipers and the volume calculated as tumor length×tumor width²/2. 30 days after implantation, mice bearing tumors between 150 and 250mm³ are randomized into two groups of six and treated with 100 mg/kg BID AZD3965 in 0.5% hydroxypropyl methyl cellulose, 0.1% tween 80 or vehicle only by oral gavage for 21 days. Measurements are continued 3 times a week for the duration of drug treatment to assess tumor growth kinetics. At sacrifice tumors are collected to determine intra-tumor lactate concentration.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Bola BM, et al. Inhibition of monocarboxylate transporter-1 (MCT1) by AZD3965 enhances radiosensitivity by reducing lactate transport. Mol Cancer Ther. 2014 Dec;13(12):2805-16.  [Content Brief]

  [2]. Polanski R, et al. Activity of the monocarboxylate transporter 1 inhibitor AZD3965 in small cell lung cancer. Clin Cancer Res. 2014 Feb 15;20(4):926-37.  [Content Brief]