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)

LNA-i-miR-221 antiproliferative activity and target silencing in retrieved MM xenografted tumors.

<p>A) q-RT-PCR of p27Kip1 mRNA levels in treated tumors retrieved from mice after intravenous LNA-i-miR-221 treatment. Raw Ct values were normalized to GAPDH housekeeping mRNA and expressed as ΔΔCt values calculated using the comparative cross threshold method (miRNA expression in LNA-i-miR-NC treated animals) ±SD. B) Western blot analysis of p27Kip1 protein in retrieved tumors from mice treated with LNA-i-miR-221 inhibitors or LNA-i-miR-NC. GAPDH was the protein loading control. C) H&E (200-fold magnification), p27Kip1 (200-fold magnification) and Ki-67 (10–fold magnification) immunohistochemistry staining of xenografted tumors retrieved from treated animals. Representative images are shown.</p

Molecular effects induced by LNA-i-miR-221 transfection in MM cells.

<p>miR-221(A) q-RT-PCR 24, 48 and 72 hours after transfection with LNA-i-miR-221 and LNA-i-miR-NC in NCI-H929 cells. The results are shown as miRNA expression levels after normalization with RNU44 and ΔΔCt calculations. Data represent the average of 3 independent experiments ±SD. B) q-RT-PCR of p27Kip1 mRNA expression 24 and 48 hours after transfection with LNA-i-miR-221 or scrambled control in NCI-H929 cells. Data represent the average of 3 independent experiments ±SD after normalization with GAPDH mRNA and ΔΔCt calculations. (*) P<0.05, (**) P<0.01. C) Western blot analysis of p27Kip1 protein in NCI-H929 cells 24, 48 and 72 hours after transfection with LNA-i-miR-221 or control. GAPDH was used as protein loading control.</p

LNA-i-miR-221 specifically recognizes the miR-221 complementary sequence.

<p>Luciferase reporter assay of NCI-H929 cells co-transfected with pLightSwitch_3′UTR Reporter Vector containing the miR-221-3p synthetic sequence cloned downstream of the Renilla luminescent reporter gene (RenSP) and miR-221/222 mimics (A) or LNA-i-miR-221 (B) or scrambled sequences as control. The firefly luciferase activity was normalized to Renilla luciferase activity. The data are shown as relative luciferase activity of miR-221/222-transfected cells versus the control (miR-NC) or LNA-i-miR-221-transfected cells versus the scrambled control (LNA-i-miR-NC). C) Dual-luciferase assay of NCI-H929 cells co-transfected with firefly luciferase constructs containing the 3′UTR of p27Kip1 and LNA-i-miR-221 or scrambled oligonucleotides (NC) as indicated. Firefly luciferase activity was normalized to r Renilla luciferase activity. The data are shown as relative luciferase activity of LNA-i-miR-221-transfected cells versus the control (NC).</p