Association between the presence of CTCs before starting a new line of chemotherapy and response to treatment.

<p>Higher response rate is observed in CTC-negative patients at baseline (A). Dynamic variation of CTCs numbers before and after treatment in patients (n = 10) with at least two determinations and at least one CTC at any time point. CTCs changes did not correlate with tumor response (B).</p

Kaplan-Meier estimates of overall survival according to a combined risk factors model with Argiris factors and CTCs.

<p>Continuous line indicates absence of both risk factors; small dotted line indicates the presence of only one of the two risk factors; large dotted line indicates the presence of both risk factors.</p

Example of CTCs analysis in a patient with mediastinal and axillary nodal metastases from an oropharyngeal squamous cell carcinoma.

<p>(A) the CellSearch output of baseline CTC analysis showing two CTCs with heterogeneous EGFR expression. (B) Timeline of CTC analysis and treatments. (C) Correlative imaging analysis by CT/PET at baseline and after chemotherapy. In this patient 3 CTCs were detected at baseline. After 4 cycles of a chemotherapy, CTC number rised to 9 suggesting progressive disease then confirmed by CT/PET imaging.</p

Patients characteristics at baseline.

<p>Patients characteristics at baseline.</p

Univariate associations between CTCs at baseline and clinico-pathologic characteristics.

<p>Univariate associations between CTCs at baseline and clinico-pathologic characteristics.</p

Clonogenic assays of CB-CD34+ cells.

<p>Results of statistical analysis of methylcellulose-based clonogenic assay performed on CB-CD34+ cells plated after 14 days of co-culture with human MSCs. (A) CFU-GEMM count; (B) BFU-E count; (C) CFU-GM count. Values are reported as mean ± SD. The results derive from three independent experiments. Abbreviations: BFU-E, Burst forming unit-erythroid; CFU, Colony forming unit; E, erythrocyte; GM, granulocyte-monocyte; GEMM, granulocyte-erythrocyte-monocyte-megakaryocyte.</p

<i>Ex vivo</i> expansion of CB-CD34+ cells with MSCs.

<p>5×10⁵ CB-CD34+ cells were cultured alone or in presence of a layer of MSCs for 10 days; the fold increase in total cell number was calculated from the original CD34+ cells seeded at day 0. The absolute number of CB-CD34+ cells was measured by flow cytometry. Values came from 5 independent replicates. Abbreviation: CB-alone: number of CB-CD34+ cells after 10 days of single culture; SN-fraction: CB-CD34+ cells in the supernatant (SN-fraction) of the co-cultures with MSCs; AD-fraction: CB-CD34+ cells grown directly in contact with MSCs layer; Total SN+AD: total number of CB-CD34+ cells after co-culture with MSCs.</p

Experimental design of the study.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172430#sec002" target="_blank">Material and method</a> section for a complete description of the figure. Ten days of culture were chosen for 2 reasons: first of all, the majority of research articles dealing with MSCs and HSCs range from 7 to 14 days of co-culturing. Secondly, 10 days of co-culturing allow to see a biological effect of MSCs on HSCs without inducing cellular senescence in stromal cells. Indeed, co-cultures were maintained in serum-free medium supplemented with Stem Cell Factor (SCF), Thrombopoietin (TPO) and Granulocyte Colony Stimulating Factor (G-CSF) (see below for details). When MSCs are cultured in this medium for more than 14 days they undergo changes in cell morphology and they begin losing their phenotypic characteristics (data not shown).</p

Gene expression profiles analysis of MSCs after co-culturing with CB-CD34+ cells.

<p>(A) Supervised analysis of MSCs-alone vs. MSCs after 10 days of co-culture with CB-CD34+ cells. A heat map of the genes differentially expressed in a statistically significant manner after co-culture (corrected p value <0.05) is plotted. (B) Gene Set Enrichment Analysis (GSEA) of MSCs-alone vs. MSCs maintained for 10 days in co-culture with CB-CD34+ cells. Top 10 gene sets from hallmark gene sets, canonical/KEGG pathways and oncogenic signatures are plotted. Please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172430#pone.0172430.s013" target="_blank">S4 Table</a> for the complete GSEA details.</p

Real-Time Quantitative Polymerase Chain Reaction PCR (qRT-PCR) analysis of <i>CCNA1</i> gene.

<p>Expression level of <i>CCNA1</i> gene was assessed by qRT-PCR. Quantification of mRNA relative quantity was calculated as relative changes in gene expression of the target gene normalised to the <i>GAPDH</i> endogenous control and relative to a calibrator sample. The values obtained were represented as relative quantity of mRNA level variations.</p