Phenotypic, Genomic, and Transcriptomic Heterogeneity in a Pancreatic Cancer Cell Line

Abstract

Objective: To evaluate the suitability of the MIA PaCa-2 cell line for studying pancreatic cancer intratumor heterogeneity, we aim to further characterize the nature of MIA PaCa-2 cells' phenotypic, genomic, and transcriptomic heterogeneity.

Materials and methods: MIA PaCa-2 single-cell clones were established through flow cytometry. For the phenotypic study, we quantified the cellular morphology, proliferation rate, migration potential, and drug sensitivity of the clones. The chromosome copy number and transcriptomic profiles were quantified using SNPa and RNA-seq, respectively.

Results: Four MIA PaCa-2 clones showed distinctive phenotypes, with differences in cellular morphology, proliferation rate, migration potential, and drug sensitivity. We also observed a degree of genomic variations between these clones in form of chromosome copy number alterations and single nucleotide variations, suggesting the genomic heterogeneity of the population, and the intrinsic genomic instability of MIA PaCa-2 cells. Lastly, transcriptomic analysis of the clones also revealed gene expression profile differences between the clones, including the uniquely regulated ITGAV , which dictates the morphology of MIA PaCa-2 clones.

Conclusions: MIA PaCa-2 is comprised of cells with distinctive phenotypes, heterogeneous genomes, and differential transcriptomic profiles, suggesting its suitability as a model to study the underlying mechanisms behind pancreatic cancer heterogeneity.

Figure 1.. Morphological heterogeneity of MIA PaCa-2 clones.

(A) Pie chart showing the proportion of MIA PaCa-2 single-cell clones based on their cellular morphology. (B) Representative images of the four clones with distinctive cellular morphology (scale bar = 50 μm). (C) Three cell morphometric parameters (cell area, circularity, and aspect ratio) were analyzed by ImageJ, showing significant morphological differences between the clones (N = 3 experiments, one-way ANOVA, ¹p < 0.05 vs. clone #1, ²p < 0.05 vs. clone #2, ³p < 0.05 vs. clone #3, ⁴p < 0.05 vs. clone #4).

Figure 2.. Differential proliferation, migration rate, and drug sensitivity of MIA PaCa-2 clones.

(A) Representative images of the MIA PaCa-2 clone cells on day 1 and day 4 after seeding (scale bar = 100 μm). (B) The plot of cell numbers of different MIA PaCa-2 clones over a 4-day culture period. The cell numbers were normalized to the cell number of day 1 in each of the clones (N = 3 experiments, one-way ANOVA, ¹p < 0.05 vs. clone #1, ²p < 0.05 vs. clone #2, ³p < 0.05 vs. clone #3, ⁴p < 0.05 vs. clone #4). (C) Representative images of the scratch wound healing assay of the four MIA PaCa-2 clones at zero and 48 hours after the scratch (scale bar = 300 μm). (D) The plot of the relative wound closure of the different MIA PaCa-2 clones at 48 hours (N = 3 experiments, one-way ANOVA, ¹p < 0.05 vs. clone #1, ²p < 0.05 vs. clone #2, ³p < 0.05 vs. clone #3, ⁴p < 0.05 vs. clone #4). (E) MIA PaCa-2 clones #1, #2, #3, and #4 were treated with gemcitabine for 72 hours and the cell numbers were measured using a luminescence cell viability assay. The cell number data was transformed into the normalized growth rate through the online GR calculator. (i) Curve fitting was performed on the normalized growth rate data to derive the dose-response curves and the corresponding GEC50 and GRinf (N = 3 experiments, n = 2–3 wells per gemcitabine concentration). (ii and iii) The GEC50 of clone #4 is statistically lower than the other clones, while the GRinf of clone #1 is statiscally higher than other clones. (N = 3 experiments, one-way ANOVA, ¹p < 0.05 vs. clone #1, ²p < 0.05 vs. clone #2, ³p < 0.05 vs. clone #3, ⁴p < 0.05 vs. clone #4).

Figure 3.. Genomic heterogeneity between the MIA PaCa-2 clones and their stability with culture.

(A) Genomic DNA of the MIA PaCa-2 parental bulk and the single-cell clones were subjected to SNPa and the resulting data were used to derive chromosome copy numbers of the clones within 1 Mb binning windows. The heatmaps illustrate chromosome copy numbers across the whole genome, where blue indicates chromosome loss (<2) and red indicates chromosome gain (>2). (B) The copy number variations (CNVs) between the clones and the parental bulk were derived by dividing the copy number of each clone by the parental bulk copy number. CNVs that are unique to a clone were observed across the genome, suggesting genomic heterogeneity between the clones. (C) Secondary single-cell clones were derived from the primary clones #1, #2, #3, and #4, and were subjected to SNPa to derive their chromosome copy number profiles. In general, most of the secondary clones clustered together based on their respective primary clones, suggesting the genomic stability of these clones with culture. However, some differences in copy number are also observed between the secondary clones derived from the same primary clone, indicating some level of plasticity in the genome over time, even though it is less than the differences observed between the primary clones. (D) The SNP sequence data was used to derive the single nucleotide variations (SNVs) between the primary clones, followed by the construction of an unrooted phylogenetic tree by using the Neighbor-Joining algorithm. The tree visualizes the genomic heterogeneity between the primary clones at the single nucleotide level, covering the distance of around 15% of the total detectable SNPs (~100,000 out of the 759,993 SNPs). The black lines are the branches of the tree, while the scale bar represents the branch length of 5% SNP mismatch. The colored circles represent the nodes of the primary clones. (E) A phylogenetic tree was constructed from the SNVs between the secondary clones. Similar to the CNVs, most of the secondary clones cluster together based on their respective primary clones, but, some degree of SNVs between the secondary clones derived from the same primary clone are also observed. The black lines are the branches of the tree, while the scale bar represents the branch distance of 2% SNP mismatch. The colored circles represent the nodes of the secondary clones. The light gray lines are guidelines to match the terminal nodes with their corresponding clone number.

Figure 4.. Differential gene expressions between the MIA PaCa-2 clones.

The primary clones #1, #2, #3, and #4 were subjected to RNA-seq, and to identify the uniquely regulated genes, differential expression analyses were performed between a given clone against the other three clones, such as clone #1 vs. clones #2, #3, and #4. A gene is considered to be uniquely regulated when it is regulated in the same direction (upregulated or downregulated) within all of the three comparisons. (A) The bar plot shows the number of genes that are uniquely regulated in each of the clones. (B) Heatmap showing normalized read counts of the uniquely regulated genes. (C-F) The uniquely regulated genes were subjected to gene set enrichment analysis using Enrichr and the top 10 statistically significant gene sets were selected (FDR < 0.05). The bar plot shows the “hallmark” gene sets that are enriched by the uniquely upregulated (red) and downregulated (blue) genes of each clone. The dashed line on the bar plots indicates the FDR cut-off.

Figure 5.. ITGAV dictates the cellular morphology of MIA PaCa-2 clones.

(A) ITGAV read counts from the RNA-seq of the MIA PaCa-2 clones (normalized to counts per million reads, CPM), showing clone #4 had significantly decreased expression of ITGAV (one-way ANOVA, ¹p < 0.05 vs. clone #1, ²p < 0.05 vs. clone #2, ³p < 0.05 vs. clone #3, ⁴p < 0.05 vs. clone #4). (B) Western blot of the ITGAV protein in the MIA PaCa-2 clones (upper band) and the β-Actin protein as the loading control (lower band) (left). Quantification of the ITGAV protein level, normalized to β-Actin, showing clone #4 had a significantly lower level of the ITGAV protein (one-way ANOVA, ¹p < 0.05 vs. clone #1, ²p < 0.05 vs. clone #2, ³p < 0.05 vs. clone #3, ⁴p < 0.05 vs. clone #4). (C and D) Representative images of the MIA PaCa-2 clone #4 and clone #4 transiently transfected with mApple-ITGAV, showing that clone #4 cells were elongated when transfected with ITGAV (mApple positive) (scale bar = 50 μm). (E) Three cell morphometric parameters (cell area, circularity, and aspect ratio) were analyzed by ImageJ, showing significant differences between clone #4 and clone #4 expressing mApple-ITGAV (Welch’s t-test, p < 0.05 vs. clone #4). (F) Western blot showing clone #1 upon knockout of ITGAV significantly reduced the ITGAV protein level (normalized to β-Actin, Welch’s t-test, p < 0.05 vs. Ctrl). (G and H) Representative images of the MIA PaCa-2 clone #1 and clone #1 transduced with a CRISPR vector targeting ITGAV, showing that the majority of clone #1 cells upon depletion of ITGAV changed from elongated to rounded morphology (scale bar = 100 μm). (I) Three cell morphometric parameters (cell area, circularity, and aspect ratio) were analyzed by ImageJ, showing significant differences between clone #1 and clone #1 with the depletion of ITGAV (Welch’s t-test, p < 0.05 vs. clone #1).