r complete data refer to S2 Fig and S3 Fig ELISA Hsp90 and flow cytometry hCD45+ data were transformed to log10 and analyzed by Pearson’s correlation. Correlations between ALL in peripheral blood and in the different tissues are shown for comparisons. Dotted line represents cut-off values for ALL detection by flow cytometry or Hsp90 levels. Data points correspond to individual samples. Numbers near each data point represent time point of sampling. PB; peripheral blood. BM; bone marrow. Circles, BCP-ALL. Triangles, T-ALL. doi:10.1371/journal.pone.0129298.g003 treated every morning, Monday to CP 868596 web Friday and on Friday afternoon; blood was collected for ELISA and flow cytometry analysis. Separate experiments were run for each of the three biomarkers. As shown in Fig 4, levels of Hsp90 were proportional to the percentage of ALL cells in peripheral blood, independently of treatment and B2M and IGFBP2 were not influenced by the PI3K inhibitor. Contrarily, production of both B2M and IGFBP2 by ALL cells in animals under dexamethasone treatment was lower than expected; indicating that decreases in circulating B2M and IGFBP2 levels following glucocorticoids treatment may not always reflect a corresponding reduction in leukemia cell numbers. This finding was confirmed with four other primary ALL. Engrafted animals were treated with dexamethasone after reaching 0.5% ALL in peripheral blood. Hsp90 levels were assessed and compared with percentage of ALL in peripheral blood during 3 weeks. For all ALL samples the correlation between Hsp90 levels and percentage of ALL cells in peripheral blood were highly significant. Results for one of the BCP-ALL 8 / 13 Plasma Hsp90 for In Vivo Monitoring of ALL Fig 4. Chemotherapy effects on biomarkers levels. Plasma biomarkers levels and matched percentage of ALL cells in peripheral blood from animals under chemotherapy treatment. Samples were collected for analysis at the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19709857 beginning, middle and end of treatment. Colored bars represent mean SD of biomarker levels for individual mice samples analyzed in duplicate. Different colors represent different treatments. The PI3K inhibitor used was AS605240. Black bars represent percentages of ALL cells in peripheral blood. The dotted line in the Hsp90 graphic represents the ELISA cut-off value. Red arrows highlight lower than expected ELISA values, when compared to levels found in untreated animals with similar percentage of ALL cells. doi:10.1371/journal.pone.0129298.g004 9 / 13 Plasma Hsp90 for In Vivo Monitoring of ALL Fig 5. Kinetic of Hsp90 levels and percentage of peripheral blood ALL cells for BCP-ALL#3. Two mice were transplanted with primary BCP-ALL cells and assessed for peripheral blood hCD45+ cell percentage by flow cytometry and Hsp90 levels by ELISA. The animals were treated daily with dexamethasone after the seventh week of transplantation. Red colored lines and symbols represent Hsp90 levels. Black lines and symbols represent the percentage of ALL cells in peripheral blood. There was no sampling in the seventh week. After the eighth week, peripheral blood samples were collected every three days. doi:10.1371/journal.pone.0129298.g005 are shown in Fig 5, to illustrate how well Hsp90 levels correlate with the percentage of ALL cells in peripheral blood both before and during treatment of animals. Furthermore, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19710274 it shows the usefulness of Hsp90 measurement for the earlier detection of ALL engraftment. Although further experiments are needed to evaluate the effect of ot