Table 1.
Primer sequences used for real time PCR analysis.
Figure 1.
In vitro establishment of KC and KPC cell lines.
(A) Inverted microscope images (4X) of UN-KC-6141, UN-KPC-960, and UN-KPC-961 PDAC cell lines after 35 passages. The three cell lines displayed typical cobblestone epithelial morphology. Genetic sequence analysis of (B) KrasG12D and (C) Trp53R172H mutation in mouse PC cell lines. Blue shaded areas represent the codon where the mutation is located. A yellow rectangle indicate the nucleotide responsible for the mutation. A pancreas from a Pdx1-Cre mouse (mouse tag UN-KPC-1207) was used as a negative control for Kras mutation.
Figure 2.
Growth kinetics of mouse PDAC cell lines.
(A) Cells were seeded in quadruplicate wells of 96-well plates and their growth was followed every day by measuring absorbance after incubation with WST-1 reagent. Data is represented as the mean absorbance (λSample = 450 nm, λRef = 600 nm) of four replicates ± standard error. Statistics were calculated in comparison to the growth curve for Panc02 (*p < 0.01, **p < 0.001, ***p < 0.0001). (B) Doubling time (TD) of cell population was calculated using the equation TD = (0.693t)/ln(Nt/N0) where t = time difference in h during log phase, Nt = absorbance value at time t, and N0 = absorbance value at initial time.
Figure 3.
Ductal characteristics of KC and KPC cell lines.
(A) Real-time PCR analysis of Amylase and CK19 in mouse PDAC cells. These mRNA transcripts were compared relative to the mRNA levels in normal pancreas. The data represents the mean fold increase of three replicates ± standard error. Statistics were calculated in comparison to normal pancreas (*p < 0.005, **p < 0.0001). (B) The protein expression of Amylase and CK19 were evaluated on mouse PDAC cell lines by confocal analysis. Amylase was visualized after staining with a secondary antibody conjugated to Alexa Fluor® 568 (Red Fluorescent) and CK19 was visualized after staining with Alexa Fluor® 488 (Green Fluorescent). Cell nuclei were stained with DAPI.
Figure 4.
Epithelial-mesenchymal characteristics of KC and KPC cell lines.
(A) Confocal microscopy images of E-cadherin (Alexa Fluor® 568, Red Fluorescent) expression in mouse cell lines. Cell nuclei were stained with DAPI. (B) Western blot analysis of E-cadherin in mouse cell lines. Protein lysates were resolved by 10% SDS-PAGE. β-actin was used as loading control. (C) Confocal microsocopy images of N-cadherin (Alexa Fluor® 488, Green Fluorescent) expression in mouse cell lines. Cell nuclei were stained with DAPI. (D) Western blot analysis of N-cadherin in mouse cell lines. Protein lysates were resolved by 10% SDS-PAGE. β-actin was used as loading control.
Figure 5.
Mucin expression in mouse PDAC cell lines.
(A) Real time PCR analysis of Muc1 and Muc4 in mouse PDAC cell lines. The mRNA transcripts were normalized to mRNA levels in the mouse fibroblast cell line NIH3T3. The data represents the mean fold increase of three replicates ± standard error. Statistical significances were calculated in comparison to NIH3T3 (*p < 0.05, **p < 0.001, ***p < 0.0001). (B) Western blot analysis of Muc4 in mouse cell lines. Protein lysates for Muc4 analysis were resolved by 2% SDS agarose gels. β-actin was used as a loading control and it was resolved in 10% SDS-PAGE. (C) Confocal microscopy images of Muc1 (Alexa Fluor® 568, Red Fluorescent) expression in mouse cell lines. Cell nuclei were stained with DAPI.
Figure 6.
Cytotoxic effects of Gemcitabine in mouse PDAC cell lines.
(A) Gemcitabine cytotoxicity in mouse PDAC cell lines was determined by the MTT cytotoxic assay. Cells were seeded in quadruplicate wells and incubated with different concentrations of Gemcitabine (100 nM–100 µM) for 48 h. After replacing media with the MTT reagent and dissolving the formazan crystals with DMSO, cytotoxicity was calculated based on the absorbance values (λ = 540nm) in cells treated with media only. The presented data are average of cytotoxicities in quadruplicate wells ± standard error. Statistical significance was calculated in comparison to Panc02 (*p < 0.01, **p < 0.001, ***p < 0.0001). (B) The half maximal inhibitory concentration (IC-50) of Gemcitabine in each cell line was determined after interpolation in the graphs of %Cytotoxicity vs. Concentration.
Figure 7.
Tumorigenicity of KC and KPC cell lines.
(A) The tumorigenic properties of UN-KC-6141, UN-KPC-960, and UN-KPC-961 cells were evaluated after orthotopic (OT) (N = 7) implantation of 1×106 cells in the respective mice background. Subcutaneous (SC) injections of UN-KPC-961 cells (5×106 cells) were also performed. OT tumors were grown for different time intervals: one month for UN-KC-6141 and two months for UN-KPC-960 and UN-KPC-961. SC tumors were grown for three weeks. Data is represented as the average weight of pancreatic tumors ± standard error. Differences in tumor size after mice euthanization were not statistically significant. (B) Hematoxylin & eosin stained tumor sections (10X) from UN-KPC-961 cells after OT implantation (left) and SC implantation (right). The tumors presented characteristics of poorly differentiated adenocarcinoma.
Table 2.
Summary of protein expression in murine PDAC cell lines.