Additional file 5: of Next-generation sequencing analysis of receptor-type tyrosine kinase genes in surgically resected colon cancer: identification of gain-of-function mutations in the RET proto-oncogene

Table S5. List of mutations identified in CC20 after the next generation sequencing analysis pipeline. (XLSX 17 kb

Additional file 3: of Next-generation sequencing analysis of receptor-type tyrosine kinase genes in surgically resected colon cancer: identification of gain-of-function mutations in the RET proto-oncogene

Table S3. List of mutated RTKs in each patient included in the study. (XLSX 11 kb

Additional file 1: of Next-generation sequencing analysis of receptor-type tyrosine kinase genes in surgically resected colon cancer: identification of gain-of-function mutations in the RET proto-oncogene

Table S1. Clinical-pathological characteristics of the 37 colon cancer patients included in the study. (XLSX 12 kb

Additional file 5: of Next-generation sequencing analysis of receptor-type tyrosine kinase genes in surgically resected colon cancer: identification of gain-of-function mutations in the RET proto-oncogene

Table S5. List of mutations identified in CC20 after the next generation sequencing analysis pipeline. (XLSX 17 kb

Additional file 4: of Next-generation sequencing analysis of receptor-type tyrosine kinase genes in surgically resected colon cancer: identification of gain-of-function mutations in the RET proto-oncogene

Table S4. Sequencing metrics of the identified RET mutations. (XLSX 10 kb

Generation of transgenic <i>R26-AKT1</i>^<i>E17K</i> mouse.

<p><b>A.</b> Schematic representation of the targeting construct used for the conditional knock-in in the <i>R26</i> locus. The human <i>AKT1</i>^<i>E17K</i> cDNA preceded by a loxP-flanked transcriptional stop cassette, was recombined into the R26 locus. Cre-mediated removal of the stop cassette links Rosa26 exon 1 to the exogenous cDNA allowing expression of the transgene. <b>B.</b> Southern blot of EcoRV digested genomic DNA derived from mESCs transfected with the targeting construct carrying mutant AKT1. Lane 1: DNA from non-targeted mESCs, lane 2 and 3: DNA from DNA from two different <i>R26-AKT1</i>^<i>E17K</i> mESCs strains. Endogenous allele corresponds to the 11.5 kb band (<i>WT</i>); mutant allele corresponds to the 4.3 kb band (<i>REC</i>). <b>C.</b> Relative mRNA expression of human AKT1^E17K by Q-RT-PCR in targeted mESCs and in the corresponding cells transfected with the Flp (pFlpE-IRES-Puro) or Cre recombinase (pCre-IRES-Puro). Data are from replicate experiments as the mean±SD. ***p<0.001. <b>D</b>. Genotype analysis by PCR on tail-tip DNA of genetically modified mice as indicated.</p

Mutant AKT1^E17K accelerates tumor formation induced by chemical carcinogens.

<p><b>A.</b> Lung tumor multiplicity in <i>R26-AKT1</i>^<i>E17K</i> infected with Ad-Cre (6 and 9 months after the treatment, respectively). Each point represents one mouse; bars represent means ± SD. **p<0.01, *p<0.05. <b>B.</b> Diameter of lung tumors generated by urethane in <i>R26-AKT1</i>^<i>E17K</i> mice treated with increasing doses of Ad-Cre. Bars represent mean diameter of tumor ±SD. *p<0.05. <b>C, D.</b> Representative H&E staining of lung lesions developed in <i>R26-AKT1</i>^<i>E17K</i> mice treated with solvent or Ad-Cre, as indicated, 9 months after urethane administration. <b>E, F.</b> Phosporylated AKT staining of lung lesions developed in <i>R26-AKT1</i>^<i>E17K</i> mice treated with solvent or Ad-Cre, as indicated, 9 months after urethane administration. Magnification as indicated.</p

MOESM1 of Ex-vivo characterization of circulating colon cancer cells distinguished in stem and differentiated subset provides useful biomarker for personalized metastatic risk assessment

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)

Immunostaining analysis of <i>R26-AKT1</i>^<i>E17K</i> mice.

<p>pAKT immunostaining of lung tissues from <i>R26-AKT1</i>^<i>E17K</i> mice treated with solvent alone (A) or infected with Ad-Cre after 9 and 18 months from infection, respectively (B, C).</p

Indirect immunofluorescence for CC10 and SP-C in mouse lung.

<p>Representative immunofluorescence images of lung sections from <i>R26-AKT1</i>^<i>E17K</i> infected with Ad-Cre, treated with urethane or both. Panels A–C show lung sections analyzed using antibodies to CC10. Panels D–F show lung sections analyzed using antibodies to SP-C. Nuclei were stained with DAPI and are shown in blue. Magnification, 60X.</p