Exosomal lipids induce human pancreatic tumoral MiaPaCa-2 cells resistance through the CXCR4-SDF-1α signaling axis

Abstract

We previously reported that exosomes secreted by human pancreatic tumor cells induce cell death through the inhibition of the Notch-1 survival pathway (Ristorcelli et al., 2009). We demonstrated that exosomal lipids evoked apoptosis of human pancreatic cancer SOJ-6 cells. Based on the lipid composition of efficient exosomes we designed Synthetic Exosome-Like Nanoparticles (SELN) in which the ratio ordered lipids versus disordered lipids was equal to 6.0 (SELN6.0). These SELN decreased SOJ-6 cells survival by inhibiting the Notch-1 pathway. However MiaPaCa-2 cells were resistant to exosomes (Ristorcelli et al., 2008) and to SELN6.0 (Beloribi et al.,2012). In this paper we aimed at deciphering the reason(s) of this resistance. We observed, in presence of SELN6.0, that the expression of the Notch IntraCytoplasmic Domain (NICD) decreases in MiaPaCa-2 cells but neither Hes-1, the nuclear target of NICD, nor the ratio Bax/Bcl-2 were affected. We further showed that in MiaPaCa-2 cells SELN6.0 induced the activation of NF-kB, which promotes the expression and the secretion of SDF-1α. This chemokine interacts with its receptor CXCR4 on MiaPaCa-2 cells and activates the Akt survival pathway protecting cells from death. This activation process promoted by exosomal lipids could have implications in tumor progression and drug resistance.

Keywords: CXCR4-SDF-1α survival axis; Exosomes; cancer cells; lipid rafts; pancreatic neoplasm.

Figure 1. Effects of SELN6.0 on the Notch pathway in MiaPaCa-2 cells

MiaPaCa-2 cells were grown until 60-70 % confluence and starved 24h prior incubation with SELN6.0 or PBS (control). At each time supernatants were removed, cells were lysed, centrifuged 30 min at 12000g to obtain proteins. 80 μg of proteins were loaded for electrophoresis and transferred onto a nitrocellulose membrane. After saturation, the membrane has been incubated overnight with the primary antibody as indicated, then washed and incubated with the required secondary POD antibody before detection. Variations were corrected for actin. (Mean +/− SD of 3 independent experiments).

Figure 2. Effects of SELN6.0 on the NF-kB signaling

MiaPaCa-2 cells were grown until 60-70% confluence and starved 24h prior incubation with SELN6.0 or PBS (control). At each time supernatants were removed, cells were lysed, centrifuged 30 min at 12 000g to obtain proteins. 80 μg of proteins were loaded for electrophoresis and transferred onto a nitrocellulose membrane. After saturation, the membrane has been incubated overnight with the primary antibody to Ser176/180-phosphorylated p-IKK (A) and to Ser32-phosphorylated IkB (B) and to Ser536 NF-kB (C) as indicated, then washed and incubated with the required secondary POD antibody before detection. Values were corrected for actin (A, B) or for total NF-kB (C). (Mean +/− SD of 3 independent experiments).

Figure 3. Effects of SELN6.0 on the phosphorylated NF-kB nuclear translocation

MiaPaCa-2 cells were seeded on 1.2 cm-diameter cover slips in 12-well plate, once adherent cells were incubated with N-Rh-PE labelled SELN6.0 for 0, 24 and 48h (red dots). At the end of the incubation time cells were washed with PBS and then fixed. Cells were permeabilized with 0.1 % saponine-PBS during 30 min at room temperature, then cells were saturated (4% BSA 0,1% saponine in PBS) and were incubated with primary antibody to phosphorylated NF-kB, during 90 min. After washes cells were incubated with Rabbit Alexa Fluor 488 secondary antibody (green dots) and nuclei were blue-colored with Draq5 at room temperature for 30 min. (Scale bar = 500 μm). Graph represents the counting of 6 areas randomly selected from independent experiments. Between 300 and 450 total cells were counted and green dots containing nuclei were reported to total nuclei number. Data are mean +/− SD.

Figure 4. Expression of Stromal Derived Factor (SDF-1α) by MiaPaCa-2 cells

A : MiaPaCa-2 cells were grown until 60-70 % confluence and starved 24h prior incubation with SELN6.0 or PBS (control). At each time supernatants were removed, cells were lysed, centrifuged 30 min at 12 000g to obtain proteins. 80 μg of proteins were loaded for electrophoresis and transferred onto a nitrocellulose membrane. After saturation, the membrane has been incubated overnight with the primary antibody to SDF-1α then washed and incubated with the secondary POD antibody before detection and variations were corrected for control and reported to actin. (Mean +/− SD of 4 independent experiments). B : Secretion of SDF-1α in MiaPaCa-2 cells conditioned medium. MiaPaCa-2 cells were grown until 60% confluence and starved 24h prior incubation with SELN6.0 (full column) or PBS only (control, empty column). At indicated time supernatants were removed, dialyzed against water, lyophilized, dissolved back in 1.6 ml distilled water and precipitated. The pellet was taken back in water, diluted in TS/TD and 35 μl were loaded for electrophoresis and transferred onto a nitrocellulose membrane. After saturation, the membrane has been incubated overnight with antibodies to SDF-1α then washed and incubated with the secondary POD antibody before detection and variations were corrected for extracellular protein concentration. (Mean +/− SD of 3 independent experiments).

Figure 5. SDF-1α is involved in MiaPaCa-2 cells resistance to SELN6.0

A : Effect of SDF-1α blocking antibody. MiaPaCa-2 cells were grown until 60% confluence and starved 24h prior incubation with SELN6.0 or PBS (control) during 48h. Conditioned medium were removed, dialyzed against water (overnight, 4°C, Cut-off 1 kDa) and lyophilized. Lyophilized conditioned medium were taken back into fresh medium, filtered on 0.2 μm filters and used for the culture of naïve MiaPaCa-2 cells (grown in 96-wells plates at 6 000 cells by well) in the presence of 0, 5 and 10 μg/ml SDF-1α blocking antibodies for 24h. (Mean +/− SD of 3 independent experiments based on at least 8 points measurements). B : Effect of SDF-1α specific siRNA on SDF-1α expression. MiaPaCa-2 cells were plated in 6-well tissue culture plates at a density of 3 × 10⁵ cells / well. Prior transfection, the culture medium was removed and replaced with OPTI-MEM culture medium. Cells were transfected either with the mix of SDF-1α siRNA or the control siRNA at a concentration of 100 nM. Cells were then lysed and proteins loaded for electrophoresis and transferred onto a nitrocellulose membrane. After saturation, the membrane has been incubated overnight with primary antibody to SDF-1α then washed and incubated with the secondary POD antibody before revelation. (Mean +/− SD of 3 independent experiments). C : Effect of SDF-1α specific siRNA mix on MiaPaCa-2 cells in the presence of SELN6.0. 24 h after transfection with SDF-1α or control siRNA, MiaPaCa-2 cells were seeded in 96 well plates (6 000 cells/well) as soon as cells became adherent they were starved one night. Then, 48 hours after transfection, cells were challenged with SELN6.0 during 24 hours (total time 72h after transfection). Finally cell survival has been assessed through a MTT test. (Mean +/− SD of 3 independent experiments based on at least 8 points measurements each).

Figure 6. Effects of cyclopamine, SELN6.0 and conditioned medium on MiaPaCa-2 cells survival

A: MiaPaCa-2 cells were seeded in 96 wells plates (4000 cells/well), then starved during 24h. Cells were incubated with tomatidine, cyclopamine, blocking antibodies to SDF-1α (γ-SDF) during 24h or with SELN6.0 during 48h then cyclopamine was added at t =24h with or without γ-SDF for an extra 24h incubation. At the end cell survival was assessed by MTT test. (Mean +/− SD of 3 independent experiments). B: MiaPaCa-2 cells were grown until 60-70 % confluence and starved 24h prior incubation with tomatidine or cyclopamine for 24h. At the end of the incubation time, cells were lysed, centrifuged 30 min at 12 000g to pellet cell debris. Then 80 μg of proteins present in supernatant were charged for electrophoresis and transferred onto a nitrocellulose membrane. After saturation, the membrane has been incubated with Gli-1 primary antibody overnight, then washed and incubated with the required secondary HRP antibody before detection and variations were reported to actin. (Mean +/− SD of 3 independent experiments). C: MiaPaCa-2 cells were grown in 10 cm diameter dishes until 60-70 % confluence before starvation for 24h. Then they were incubated with freshly prepared SELN6.0 (black column) or with PBS (grey column) during 48h. Media were collected, dialyzed overnight against water at 4°C and lyophilized. In parallel naïve MiaPaCa-2 cells were grown in 96 wells plates (6 000 cells/well) and starved for 24h. Then lyophilized media were diluted with fresh medium, filtered on 0.2 μm filters and used for the culture in 96 wells plates with or without cyclopamine (CPA, 0 μM, 10 μM and 20 μM). After 24h, cell survival was assessed by a MTT test. (Mean +/− SD of 3 independent experiments based on at least 12 point measurements each).

Figure 7. Expression of CXCR4 and CXCR7 by MiaPaCa-2 cells

A : MiaPaCa-2 cells were seeded on 1.2 cm-diameter cover slips in 12-wells plate, once adherent cells were seeded in appropriate medium on cover-slips in 12 well-plates. Cells were fixed (2 % paraformaldehydein PBS, 37 °C, 15 min) and saturated (4% BSA in PBS, 30 min). The cells were then incubated successively with the primary antibodies to CXCR4 or to CXCR7 for 90 min and then with secondary antibody to IgG coupled to AlexaFluor 488 for 45 min. The cell nuclei were labelled 30 min with 1 μM Draq5, a far-red fluorescent DNA dye. All the later stages were carried out at 4°C. (Scale bar = 500 μm). B: Subconfluent monolayers of MiaPaCa-2 cells were harvested and suspended in DMEM containing 10 % FCS during 30 min at 37 °C. The single cell suspension (10⁶ cells/ml) was incubated for 90 min at 4°C in the presence of antibodies to CXCR4 or to CXCR7. Cells were rinsed three times with ice-cold PBS and then incubated for 45 min at 4°C with the appropriate secondary conjugated antibody. Cell-bound fluorescence was quantified (Flowjo program). Each value represents the mean fluorescence per cell. Non-specific labeling was determined by incubating cells with the secondary antibody alone (control). Graphs are representative of tree independent experiments and data represent the mean +/− SD obtained from these separate experiments.

Figure 8. CXCR4 is in involved in MiaPaCa-2 cells resistance to SELN6.0

A : Effect of CXCR7 and CXCR4 specific siRNA on respective expression. MiaPaCa-2 cells were plated in 6-well tissue culture plates at a density of 3 × 10⁵ cells / well. Prior transfection, the culture medium was removed and replaced with OPTI-MEM culture medium. Cells were transfected either with the mix of CXCR7 or CXCR4 specific siRNA or the control siRNA at a concentration of 100-150 nM. Cells were then lysed and proteins loaded for electrophoresis and transferred onto a nitrocellulose membrane. After saturation, the membrane has been incubated overnight with primary antibody to CXCR4 and CXCR7 then washed and incubated with the secondary POD antibody before revelation. (Mean +/− SD of 3 independent experiments). B : Effect of CXCR4 and CXCR7 specific siRNA on MiaPaCa-2 cells in the presence of SELN6.0. 24 h after transfection with CXCR4 and CXCR7 specific siRNA or control siRNA, MiaPaCa-2 were seeded in 96 well plates (6 000 cells/well) as soon as cells became adherent they were starved one night. Then, 48 hours after transfection, cells were cultured in control or SELN6.0 conditioned medium (see Figure 8A) and challenged with SELN6.0 or with CPA (10 μM) during 24 hours (total time 72h after transfection). Finally cell survival has been assessed through a MTT test. (Mean +/− SD of 3 independent experiments based on at least 8 points measurements each). White column; cell survival in the presence (+) or absence (−) of conditioned medium of control cells or of SELN6.0 pre-incubated cells. Grey and black columns; cell survival in the presence (+) or absence (−) of conditioned mediums of control cells or of SELN6.0 pre-incubated cells with added SELN6.0 (grey) or with added CPA (10 μM, black).

Figure 9. Phosphorylation of Akt in MiaPaCa-2 cells upon incubation with SELN6.0

MiaPaCa-2 cells were grown until 60-70 % confluence and starved 24h prior incubation with SELN6.0 or PBS (control). At each time supernatants were removed, cells were lysed, centrifuged 30 min at 12 000g to obtain proteins. 80 μg of proteins were loaded for electrophoresis and transferred onto a nitrocellulose membrane. After saturation, the membrane has been incubated overnight with the primary antibody to total Akt, to Thr308-phosphorylated Akt (Thr308 p-Akt, A) and to Ser473-phosphorylated Akt (Ser473 p-Akt, B) as indicated, then washed and incubated with the required secondary POD antibody before detection. Values were reported to total Akt. (Mean +/− SD of 3 independent experiments).