Figure 3. Effect of PrPC-positive serum and tumor tissue on doxorubicin efficacy.

(A) Comparison of doxorubicin dose-response profiles in HM.LNm5 cells supplemented with 10% FBS or 10% serum from patients B1288 or B474. For patient serum assays, cells were first depleted of endogenous PRNP using siRNA. Serum PrP^C concentration was measured by ELISA. The assay was performed within a physiologically relevant range of doxorubicin concentrations (gray shading, 10–100 nM, 24–96 hours after infusion of 75 mg/m² dose; ref. 14). (B) PRNP-depleted HM.LNm5 cells were incubated for 72 hours with conditioned medium from SCRsi-transfected HM.LNm5 (CM) cells or media from PRNP-depleted HM.LNm5 cells supplemented with 10% human serum. Parallel cultures were incubated after specific depletion of PrP^C by immunoprecipitation (IP; 3F4 antibody). Western shows PrP^C levels (with 1% of input blotted for HSP70 as a loading control), and the bar graph shows the effect of depleting PrP^C from the media on total cell number after 72 hours growth in the presence of doxorubicin (1 μM; mean ± SEM from 3 assays). (C and D) Fluorescent imaging of doxorubicin (red) in the nuclei of PRNP-depleted HM.LNm5 cells cultured with human serum. Ten image fields were analyzed in triplicate (PRNPsi, ***P = 0.002; +B1288, *P = 0.045). Scale bar: 100 nm. (E) Correlation between patient serum PrP^C concentration (determined by ELISA) and doxorubicin or epirubicin IC₅₀ in PRNP-depleted HM.LNm5 cells. Samples were tested in duplicate. (F) Correlation between patient serum PrP^C concentration and doxorubicin or epirubicin IC₅₀ in PRNP-depleted BT549 cells. (G) Co-IP of doxorubicin or epirubicin in conditioned media of HM.LNm5 cells by PrP^C using 3F4, with fluorescent readout at 480 nm compared with control IP of beads alone. Conditioned media albumin was a loading control (mean ± SEM is shown for 3 experiments; ***P = 0.005). (H) Comparison of doxorubicin and epirubicin structures, highlighting epimerization of the sugar moiety hydroxyl group.