organism: Homo sapiens
description: Protocols: CCSCs were isolated from Fresh post-surgery human colorectal cancer tissues. Tissue samples were collected immediately after each patient's surgery, quickly washed 2-3 times in cold saline, then transferred in Dulbecco's modified Eagle's medium containing 3% penicillin-streptomycin-amphotericin B solution and kept in this medium at 4°C until processing within 24-48 hours. For tissue disaggregation, tumor samples were incubated in DMEM with 300 units/ml collagenase type I, 100ug/ml of penicillin/streptomycin, 50ug/ml gentamicin, 0.25ug/ml amphotericin B and 100 units/ml DNAse and incubated for 3-4 h at 37°C (De Angelis et al., 2016). Cells suspension was then filtered through 70-um and 40-um nylon meshes. The resulting single-cell suspension was plated in NeuroCult NS-A medium containing 20 ng/ml of EGF and 10 ng/ml of FGF2 and cultured in humidified atmosphere at 37°C, 5% O2 and 5% CO2 (Galli et al., 2004). For orthotopical tumor xenografts, 2x106 luc-CCSCs cells were pelleted and resuspended in 20ul 5:1 DMEM/Cultrex with a 30-gauge 0.3x13mm needle attached to an insulin syringe. The skin of immunocompromized SCID mice (5-6 weeks of age) was opened with forceps, the abdominal wall was cut and care was taken not to damage the abdominal organs. The cecum was identified and exposed outside the body cavity to identify the colon. The caecum was replaced in the body cavity and the colon was rehydrated with warm saline and the cells were injected in the ascending colon at 5 locations. The colon was rehydrated and returned to the body cavity. The abdominal wall was closed with Vicryl 4-0 (continuous suture) and the skin with surgical wound clips. Tumor formation and growth was indirectly calculated in mice successfully injected once a week by sequential images taken by In vivo Lumina as in (Binda et al., 2012; Binda et al., 2017) from ventral and dorsal views. Region of interest (ROI) from displayed images were identified around the tumor sites and were quantified as total flux (photons/s/cm2) using Living Image software. Mice were sacrificed at different times comprised between 5 and 9 weeks post-injection, according to the cell line originally injected. Tissue specimens from CCSCs, mCCSCs and cCCSCs-derived primary colon tumors, and from mesenteric lymph nodes, lung, liver, spleen and brain metastases were excised and processed as described in (Binda et al., 2012; Binda et al., 2017; Galli et al., 2004). CCSCs cells were isolated from fresh human colorectal cancer specimens from patients who underwent surgery. CTCs cells were isolated from CCSCs-bearing mice. mCCSCs cells were isolated from distant metastatic organs of both CCSCs- and CTCs-bearing mice. Single-cell suspension was plated in NeuroCult NS-A medium containing 20 ng/ml of epidermal growth factor (EGF) and 10 ng/ml of fibroblast growth factor (FGF2) and cultured in humidified atmosphere at 37°C, 5% O2 and 5% CO2. Genomic DNA was extracted using the Blood & Cell Culture DNA Midi Kit (Qiagen). Truseq custom amplicon sample kit, (Illumina Inc, CA, USA) for targeted capture and library preparation was prepared from 250ng of double stranded DNA accordingly to manufacturers instructions.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-06-15T04:02:31Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-06-05T16:30:50Z
attributes.tag: External Id
attributes.value: SAMEA5694130
attributes.tag: INSDC center name
attributes.value: Institute for Stem Cell Biology, Regenerative Medicine and Innovative Therapies (ISBReMIT) - IRCCS Casa Sollievo della Sofferenza
attributes.tag: INSDC first public
attributes.value: 2019-06-15T04:02:31Z
attributes.tag: INSDC last update
attributes.value: 2019-06-05T16:30:50Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6944:mCCSCs#1_lung
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: colorectal cancer stem-like metastatic cells (mCCSCs)
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: metastatic colorectal cancer
attributes.tag: growth condition
attributes.value: in mouse xenograft
attributes.tag: individual
attributes.value: Patient 1
attributes.tag: sample name
attributes.value: E-MTAB-6944:mCCSCs#1_lung
attributes.tag: sampling site
attributes.value: lung
attributes.tag: scientific_name
attributes.value: Homo sapiens