Medullary thyroid cancers (MTCs) are derived from calcitonin-producing cells (C cells) of neuroendocrine origin. Rb heterozygous mice develop low-grade C cell adenocarcinoma following biallelic inactivation of the Rb tumor suppressor gene loci. Additional inactivation of another tumor suppressor gene such as Trp53, Arf or Cdkn1a allows Rb-deficient mice to generate more aggressive C cell adenocarcinoma (Takahashi et al., 2006; Shamma et al., 2009; Kitajima et al., 2015) . To characterize C cell

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... adenocarcinoma cells derived from Rb-deficient mice of different genetic backgrounds, we attempted to extract C cell adenocarcinoma cells from primary thyroid tumor tissue. Since primary mouse small cell lung cancer (SCLC) cells those originate in neuroendocrine cells that also stems C cells, can be established both as non-adhesive and adhesive cells (Calbo et al., 2011) , we applied their method to MTCs. Here we describe our isolation technique for non-adhesive and adhesive cell cultures from primary medullary thyroid tumor tissue. We found that the molecular markers of C cell such as Calcitonin and Ascl1 are predominantly enriched in the non-adhesive population (Kitajima et al., 2015) . This is in line with the fact that one of most commonly distributed human MTC cell line TT is non-adhesive. Materials and Reagents 1. 12 well cell culture plate (Thermo Fisher Scientific, catalog number: 150628) 2. 100 mm cell culture dish (Thermo Fisher Scientific, catalog number: 172931) 3. 5 ml centrifuge tube (Thermo Fisher Scientific, catalog number: 339650) 4. Phosphate buffered saline (PBS) (pH 7.2) (Life Technologies, catalog number: 20012027