Table 1.
Patients information.
Table 2.
Overview of EpoR RT-PCR primers [24].
Table 3.
Primary antibodies.
Figure 1.
Level of sEpo but not Hb was associated with a worse prognosis in PDAC.
The differences in preoperative levels of Hb (A) and sEpo (B) in blood of donors (n = 9), CP (n = 13) and PDAC (n = 87) patients (see table 1A and 1B for full information regarding studied sub-cohorts). (C) Kaplan-Meier analysis of survival data showing that a higher level of sEpo (upper quartile in panel B, ≥16 mU/ml) was associated with shorter survival of PDAC patients; ms = median survival time, in months. (D) Reduced sEpo levels in stage I–III PDAC patients (n = 64) and their restoration in metastatic patients (n = 23). (E) Prevalence of anemia and Hb values were equally distributed during PDAC progression. (F) sEpo inversely correlated with Hb in PDAC. The numbers in panels A, B, D and E depict median levels. Statistically significant differences are labeled by asterisks: p≤0.05 (*), p<0.01(**) or p<0.001(***).
Figure 2.
Aberrant expression of Epo mRNA in pancreatic tissues and tumor cells.
(A) qRT-PCR analysis of pancreatic tissues revealed gradual loss of Epo mRNA expression in diseased pancreata. (B) Except for T3M4, low levels of Epo mRNA expression were found in pancreatic tumor cell lines exposed to or not exposed to 0.75%-hypoxia. Nx = normoxia; Hx = Hypoxia. (C) Barely detectable yet elevated Epo mRNA expression was found in pancreatic specimens obtained from stage IV PDAC patients (n = 12) compared to stage I–III (n = 92) patients. The numbers in panels A and C depict median levels. Statistically significant differences are labeled by asterisks: p≤0.05 (*), p<0.01(**) or p<0.001(***).
Figure 3.
Ectopic sources of Epo in tissues of patients with pancreatic diseases.
(A) Remnants of Epo-producing islets in degrading CP-parenchyma. (B) Epo-negativity of tumor cells in primary pancreatic lesion, except for sporadic focal staining observed in intracellular vacuoles of tumor cells (C, arrows and inset). Peripheral capillaries (C, arrowheads) and vasa vasorum (D, arrowheads) of bigger vessels frequently demonstrated Epo positivity in PDAC and CP. (E, F) In liver, cytoplasmic staining of hepatocytes was strong in areas directly bordering Epo-negative metastatic tumor cells and inflammatory infiltrates, but faded away distally, thus pointing to the spatially regulated de novo synthesis and creation of multiple Epo-rich niches. Insets in A, D and E depict negative (isotype IgG) controls; insets in C and F show high-magnification (×630) images of staining patterns in intracellular vacuoles of tumor cells and cytoplasm in hepatocytes.
Figure 4.
Detection of EpoR-positive tumor cells in PDAC pancreata.
(A) Staining of erythroid precursor cells in human bone marrow with anti-EpoR 3D10 antibody (inset, ×400) and (B) loss of staining if the anti-EpoR antibody has been pre-incubated with soluble EpoR (sol-EpoR). (C, E) Varying intensity and focal character of EpoR-immunopositivity of tumor cells in PDAC tissues (arrows) and (D) blocking of EpoR signal with sol-EpoR. (F) Sporadic EpoR-immunopositivity of non-malignant structures within a PDAC sample and blocking of EpoR signal with sol-EpoR (inset).
Figure 5.
Expression of EpoR in PDAC cells.
(A) qRT-PCR analysis revealed constitutive EpoR mRNA expression in all studied cancer cell lines and its strong up-regulation under hypoxic conditions. Nx = normoxia, Hx = hypoxia. (B) EpoR was detectable on the surface of hEpoR-transduced Ba/F3 cells and PANC-1 cells by flow cytometry analysis using an FITC-labeled antibody. Mouse IgG2b was used as a negative control. (C) Left panel: detection of the full-length EpoR protein in cell lysates of hEpoR-transduced NIH/3T3 cells and the recombinant sol-EpoR protein using the 3D10-antibody by Western blot analysis (with GAPDH as loading control). Right panel: detection of EpoR in pancreatic tumor cells and hEpoR-NIH/3T3 by immunoprecipitation with the 3D10-antibody followed by immunoblotting with the C-20 antibody.
Figure 6.
Activation of PI3K/Akt but not Jak2/STAT5 signaling in PDAC cells exposed to Epo.
(A) Phosphorylation status of Stat5 was assessed in serum-starved pancreatic tumor cells and hEpoR-transduced NIH/3T3 cells stimulated with 50 U/ml erythropoietin (Epo) for 10 min. Clear accumulation of pSTAT5 was observed in hEpoR-NIH/3T3 but not in mock-transduced NIH/3T3 cells or in PDAC cells, independent of the level of constitutive pSTAT5 activation. (B) Phosphorylation status of Akt was assessed in serum-starved (left panel) or non-starved (right panel) PANC-1 cells consequently stimulated with Epo at 0–50 U/ml for 15 min. Epo-enhanced pAkt phosporylation was detected only under conditions of serum starvation and could be specifically inhibited by anti-EpoR antibody (+Ab) or phosphatidylinositol-3-OH kinase (PI3K) inhibitor LY2940020 (+LY). (C) Accumulation of mRNA coding for soluble EpoR isoform (upper panel; primers: EpoR_S5/6, table 2) as compared to mRNAs coding for full-length isoforms (two middle panels) and further related to expression of ß-actin (lower panel). 3′-end-based detection of EpoR mRNA was performed with antisense primers binding after (EpoR_FL1/2) or prior to a stop codon (EpoR_FL3/4) as visualized by a hEpoR plasmid carrying only the coding sequence for a full-length isoform.