MOESM1 of Ex-vivo characterization of circulating colon cancer cells distinguished in stem and differentiated subset provides useful biomarker for personalized metastatic risk assessment
Creators: name: Malara, Natalia
name: Trunzo, Valentina
name: Foresta, Umberto
name: Amodio, Nicola
name: Vitis, Stefania De
name: Roveda, Laura
name: Mariagiovanna Fava
name: MariaLaura Coluccio
name: Macrì, Roberta
name: Vito, Anna Di
name: Costa, Nicola
name: Mignogna, Chiara
name: Britti, Domenico
name: Palma, Ernesto
name: Mollace, Vincenzo
Publisher: name: Figshare
Year: 2016
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: 60506 Virology

description: Additional file 1: Figure S1. Sensibility, specificity and purity of CTCs detection methodology. The sensitivity of the methodology was calculated through the formula employing mean values (expressed in percentage) for each CTCs subsets identified by the combined expression of CK20 and CD45, found in the total cellular suspension collected from the working density phase. The sensitivity or the capability to detect the real subset of CTCs CK20pos corresponded to 91 %. The specificity, corresponding to the probability of a negative test, was calculated at about 87 %. Finally, the purity was at 75 %. Figure S2. Resolution of CTCs detection methodology. To verify that the collected fraction was enriched in cancer cells, HCT 116 cells were infected with pAdenoVator-CMV-IRES-GFP reporter. Human cancer colon cell lines HCT 116 were cultured in RPMI1640 medium containing 10 % fetal bovine serum (FBS), 2 mmol/l L-glutamine, and 30 mg penicillin G/0.05 g streptomycin. Cells were plated at 8 x 106 per well onto a six-well plate 24 hours before infection, and were infected with adenoviral vector. In order to perform infections, HCT 116 cells were incubated with pAdenoVator-CMV5(CuO)-IRES-GFP (Qbiogene, Carlsbad, CA) in serum free medium for 1 hour at 37 °C. Both vectors were used at multiplicity of infection (m.o.i.) of 3000 physical particles/cell, experimentally determined as the lowest m.o.i. at which the majority of the cell population is infected (as assessed by EGFP expression). Twenty-four hours later, both adherent and floating cells were harvested, washed in PBS and counted. Different concentration of HCT 116-GFP (HCT 116*) were put in entire blood sample (5 ml) and were evaluated through cytometric analysis. The resolution for the minimal concentration of HCT 116* (8 x 103 cell/5 ml) put in a volume of peripheral blood sample of 5ml, useful to detect them in the working density phase, was calculated at 5,8 cells/5 ml (B). Figure S3. DTCs in livers of mice treated with localized and advanced cancer eCTCs. Dot Plots report the expression of CK20 antigen on human colon cancer cells disseminated within liver tissue of mouse submitted to xenograft procedure. In particular, dot plot in (A) shows human colon cancer cell CK20 positive founded in liver tissue of mouse injected with eCTCs-CXCR4negCKneg referred as control. Dot plots in (B) and (C) show human cancer colon cells expressing CK20 marker in liver tissues of mouse injected with eCTCs-CXCR4posCKpos derived from localized (B) and advanced (C) colon cancer cases respectively. Figure S4. xenograft developed with circulating stem cells. Xenograft procedure developed injecting eCTCs-CD45negCD133pos organized in spheres (A). In (B) immunofluorescence positive for CD133 (green staining). In (C) Tumour formations produced within 2 weeks and after 80 days. Immunohistochemical analysis shows the distribution of the cancer colon cells expressing CD133 (brown staining) in the tumour sections of 8 μm (D).