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. 2019 May 28;8:e40226. doi: 10.7554/eLife.40226

Figure 2. Pancreatic cancer cell exosomes function as an initiator in malignant cell transformation.

(A) Two-stage cell transformation assay shown. NIH/3T3 cells were treated with a tumor initiator for 3 days (Days 3–6) and the tumor promoter for 2 weeks (Days 8–21). After 42 days, cells are fixed with methanol and stained with Crystal Violet for malignant foci counting. (B) Representative images of stained cells showing foci formation (arrows) from untreated cells and cells treated with MCA/DMSO, DMSO/TPA, MCA/TPA, or Capan-2 exosomes (ExC)/TPA (initiator/promoter). (C) Quantification of foci formed at the end of cell transformation assays. The average foci/well were determined via double-blind counting as described in Materials and methods. The red dashed line represents the established level of background foci present in untreated cells. Initiator/promoter treatments resulting in increased foci formation above background include MCA/TPA (p=0.008) and all cancer cell-derived exosomes: ExC/TPA (p=0.0002), ExM/TPA (p<0.0001), ExP/TPA (p=0.007), and ExBx/TPA (p=0.0003). Bars shown in gray represent controls that did not result in foci formation above background. Bar shown in pink shows results from normal cell (HPDE) exosome/TPA treatment (p=0.0004). (D) Quantification of foci formed after use of a different promoter, CdCl2. CdCl2 acts as a promoter leading to increased foci formation above background when used with the initiators MCA (p<0.0001) or Capan-2 exosomes (ExC) (p<0.0001). Asterisks indicate significant differences from either control treatment or MCA/TPA treatment as determined by unpaired, two-tailed t-test with Welch’s correction (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Red (+)=initiator used; purple (+)=promoter used.

Figure 2—source data 1. Relates to Figure 2.
Quantification of foci formed from two-stage cell transformation assays shown in Figure 2C.
elife-40226-fig2-data1.xlsx (12.4KB, xlsx)
DOI: 10.7554/eLife.40226.012
Figure 2—source data 2. Relates to Figure 2.
Quantification of foci formed from two-stage cell transformation assays shown in Figure 2D.
elife-40226-fig2-data2.xlsx (9.2KB, xlsx)
DOI: 10.7554/eLife.40226.013

Figure 2.

Figure 2—figure supplement 1. Characterization of initiation activity of pancreatic cancer cell exosomes.

Figure 2—figure supplement 1.

(A) Quantification of foci formed at the end of cell transformation assays, foci were counted as described in Materials and methods. The red dashed line represents the established level of background foci present in untreated cells. Initiator/promoter treatments resulting in increased foci formation above background include MCA/TPA (p=0.008) and ExC/TPA (p=0.0002), as previously shown. Treatment of cells with primary fibroblasts exosomes (ExFB) (initiator)/TPA (promoter) did not result in increased cell transformation (pink bar, p=0.0007). Treatment of cells with ExC heated exosomes (initiator)/TPA (promoter) did not result in increased cell transformation (dashed blue bar, p=0.0024). (B) Treatment of cells with ‘pure’ ExC exosomes (fraction three from density sucrose gradient fractionation) as an initiator and TPA as a promoter did result in increased cell transformation above background (dotted blue bar, p<0.0001). (C) Dose-response studies. ExC exosomes were tested in the assay at a measured protein concentration ranging from 0.08 ng/mL to 2400 ng/mL (concentrations in ng/mL are shown in red). Asterisks indicate significant differences from either control treatment or MCA/TPA treatment as determined by unpaired, two-tailed t-test with Welch’s correction (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Red (+)=initiator used; purple (+)=promoter used.
Figure 2—figure supplement 1—source data 1. Relates to Figure 2—figure supplement 1.
Quantification of foci formed from two-stage cell transformation assays shown in Figure 2—figure supplement 1A.
elife-40226-fig2-figsupp1-data1.xlsx (9.3KB, xlsx)
DOI: 10.7554/eLife.40226.009
Figure 2—figure supplement 1—source data 2. Relates to Figure 2—figure supplement 1.
Quantification of foci formed from two-stage cell transformation assays shown in Figure 2—figure supplement 1B.
elife-40226-fig2-figsupp1-data2.xlsx (9.1KB, xlsx)
DOI: 10.7554/eLife.40226.010
Figure 2—figure supplement 1—source data 3. Relates to Figure 2—figure supplement 1.
Quantification of foci formed from two-stage cell transformation assays shown in Figure 2—figure supplement 1C.
elife-40226-fig2-figsupp1-data3.xlsx (9.5KB, xlsx)
DOI: 10.7554/eLife.40226.011