Primary mPDAC cell cultures were isolated from autochthonous PDAC and cultured as described previously59 (link)
. All cells were cultivated for less than 30 passages, authenticated by genotyping and tested for mycoplasma contamination by PCR. Conventional human PDAC cell lines and primary patient derived low passaged PDAC cell cultures were established and cultured as previously reported58 (link)
.
For long-term clonogenic cell proliferation assays, cells were seeded into 24-well plates (density of 1-2×103 cells/well, depending on growth rate. The following day, plates were treated with different concentrations of drugs as indicated. Every 7 days, media and drug were refreshed. Cells were fixed and stained with 0.2% crystal violet in an ethanol/water solution 7 to 13 days after the start of treatment, according to the confluence reached by the untreated control. Crystal violet was solubilized with 10% acetic acid and absorbance was quantified at 595 nm. The resulting values were used to calculate the Bliss synergy score with the online software Synergy Finder (v1.0)60 (link)
. All assays were performed independently at least three times. Trametinib, nintedanib, AZD-4547, imatinib and PF-3758309 were obtained from Selleckchem, 4-OHT from Sigma, murine anti PD-L1 mAb (Anti PD-L1-mIgG1e3 InvivoFit™) was purchased from InvivoGen, and tamoxifen for in vivo treatment from Sigma.
. All cells were cultivated for less than 30 passages, authenticated by genotyping and tested for mycoplasma contamination by PCR. Conventional human PDAC cell lines and primary patient derived low passaged PDAC cell cultures were established and cultured as previously reported58 (link)
.
For long-term clonogenic cell proliferation assays, cells were seeded into 24-well plates (density of 1-2×103 cells/well, depending on growth rate. The following day, plates were treated with different concentrations of drugs as indicated. Every 7 days, media and drug were refreshed. Cells were fixed and stained with 0.2% crystal violet in an ethanol/water solution 7 to 13 days after the start of treatment, according to the confluence reached by the untreated control. Crystal violet was solubilized with 10% acetic acid and absorbance was quantified at 595 nm. The resulting values were used to calculate the Bliss synergy score with the online software Synergy Finder (v1.0)60 (link)
. All assays were performed independently at least three times. Trametinib, nintedanib, AZD-4547, imatinib and PF-3758309 were obtained from Selleckchem, 4-OHT from Sigma, murine anti PD-L1 mAb (Anti PD-L1-mIgG1e3 InvivoFit™) was purchased from InvivoGen, and tamoxifen for in vivo treatment from Sigma.