Acidic Growth Conditions Promote Epithelial-to-Mesenchymal Transition to Select More Aggressive PDAC Cell Phenotypes In Vitro

Abstract

Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by an acidic microenvironment, which contributes to therapeutic failure. So far there is a lack of knowledge with respect to the role of the acidic microenvironment in the invasive process. This work aimed to study the phenotypic and genetic response of PDAC cells to acidic stress along the different stages of selection. To this end, we subjected the cells to short- and long-term acidic pressure and recovery to pH_e 7.4. This treatment aimed at mimicking PDAC edges and consequent cancer cell escape from the tumor. The impact of acidosis was assessed for cell morphology, proliferation, adhesion, migration, invasion, and epithelial-mesenchymal transition (EMT) via functional in vitro assays and RNA sequencing. Our results indicate that short acidic treatment limits growth, adhesion, invasion, and viability of PDAC cells. As the acid treatment progresses, it selects cancer cells with enhanced migration and invasion abilities induced by EMT, potentiating their metastatic potential when re-exposed to pH_e 7.4. The RNA-seq analysis of PANC-1 cells exposed to short-term acidosis and pH_e-selected recovered to pH_e 7.4 revealed distinct transcriptome rewiring. We describe an enrichment of genes relevant to proliferation, migration, EMT, and invasion in acid-selected cells. Our work clearly demonstrates that upon acidosis stress, PDAC cells acquire more invasive cell phenotypes by promoting EMT and thus paving the way for more aggressive cell phenotypes.

Keywords: EMT; PDAC; acid-selection; acidic tumor microenvironment; cell adhesion; cell invasion; cell migration; cell proliferation.

Figure 1

Effects of acidic pH_e on PANC-1 cells’ intracellular pH and PDAC cells’ morphology. (a) Scheme of the different acidic pH_e phenotypes established. Control cells were kept in physiological pH_e culture conditions (pH_e= 7.4), while acidic phenotypes were constituted by PDAC cells exposed for different periods to acidic pH_e: 4 days (cell model named “4 days pH_e 6.6”), 1 month (pH_e-selected), and 1-month long exposure followed by recovery to physiological pH_e for 2 weeks (pH_e-selected + 7.4). (b) Mean traces (± SEM) of at least 4 independent experiments illustrating the resting pH_i values in PANC-1 control cells (blue) and the different acidic phenotypes. Cells were loaded with 3 µM BCECF, and pH_i values were recorded for 3 min as a fluorescent ratio (490/440 nm) changes following exposition to pH_e 6.6 for 4 days (green), 1 month (light orange), and 1-month prior recovery to pH_e 7.4 for 2 weeks (red). (c) Quantification of pH_i values in PANC-1 control and acidic phenotypes from 3 min recording. Each dot indicates the mean value of one independent experiment (n ≥ 108 cells for each condition). Data were analyzed using the Kruskal–Wallis H-test and Dunn’s multiple comparison test, * p < 0.05, ns = not significant. (d) Alexa Fluor 488-phalloidin (F-actin, green) and DAPI (nucleus, blue) staining of the different PANC-1 (top) and Mia PaCa-2 (bottom) cell models on a 1% gelatin-coated surface. Scale bar 20 µm. (e) Quantification of cell area in µm² (left) and cell circularity index (right) of the different PANC-1 and (f) Mia PaCa-2 cell models. Data were reported as mean (± SEM) of 5 representative regions per condition; 3 independent experiments were performed for each condition. Data were analyzed using One-way ANOVA with Dunnett’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant.

Figure 2

Effects of acidic pH_e on PDAC cells’ viability and proliferation. (a) Representative fluorescence images of PANC-1 and (b) Mia PaCa-2 cell proliferation obtained by EdU staining assay (red, Alexa Fluor 647) and Hoechst (blue) nuclear staining. Scale bar = 20 µm. (c) Quantification of the percentage of PANC-1 and (d) Mia PaCa-2 EdU-positive cells upon treatment with acidic pH_e. Data were reported as the percentage of EdU/Hoechst-positive cell mean ± SEM from 4 representative regions for each condition. Time course of proliferation of the different models of (e) PANC-1 and (f) Mia PaCa-2 cells assessed by an MTS assay. Significant differences between control cells vs. all other conditions at 96 h. (g) Determination of PANC-1 (left) and Mia PaCa-2 (right) cell viability by trypan blue exclusion assay in control cells (pH_e 7.4), 4 days pH_e 6.6, pH_e-selected (1 month in pH_e 6.6) and pH_e selected + 7.4 (1 month in pH_e 6.6 followed by 2 weeks in pH_e 7.4). (h) Time course of PANC-1 (blue) and Mia PaCa-2 (yellow) cell viability by trypan blue exclusion assay in control cells exposed to acidic pH_e for different times. Significant differences in PANC-1 (*) or Mia PaCa-2 cells ($) at each time point of acidic treatment vs. 1 h. (i) Determination of PANC-1 (left) and Mia PaCa-2 (right) cell viability as assessed by trypan blue exclusion assay in control cells (pH_e 7.4) and pH_e selected + 7.4 (1 month in pH_e 6.6 followed by 2 weeks in pH_e 7.4) cells in pH_e 7.4 and following 96 h treatment in pH_e 6.6. Data were presented as mean ± SEM using One-way ANOVA with Dunnett’s multiple comparisons test. All data shown were obtained from three independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, $$, p < 0.01, $$$$ p < 0.0001, ns = not significant.

Figure 3

Effects of acidic pH_e on PDAC cell adhesion, migration, and invasion. (a) Quantification of PANC-1 and (b) Mia PaCa-2 cell adhesion assays. Cells were exposed to acidic conditions for different periods. Data were reported as mean ± SEM from at least 4 representative regions for each condition. (c) Representative brightfield microscopic images of crystal violet-stained PDAC cell (in blue) that migrated through the transwell membrane in the migration assay. Scale bar = 100 µm. Quantification of the mean number of migrated (d) PANC-1 and (e) Mia PaCa-2 cells determined in a transwell 18-h long migration assay. Data were reported as mean ± SEM. (f) Representative images of PDAC cell models that invaded through the Matrigel-coated transwell membrane in the invasion assay. Scale bar = 100 µm. Quantification of the mean number of invaded cells of the 18-h long transwell invasion assay in (g) PANC-1 and (h) Mia PaCa-2 cells. Data were reported as mean ± SEM of three independent experiments and analyzed using One-way ANOVA with Dunnett’s multiple comparisons test. All data shown were obtained from three independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Effects of a pH_e gradient on PDAC cells migration, invasion, and invadopodia activity. (a) Representative microscopic images of PDAC cells’ migration assay, showing cells that migrated through the transwell membrane in a pH gradient, allowing cells to move from an acidic compartment to the pH 7.4 bottom part of the transwell. Scale bar = 100 µm. (b) Quantification of the mean number of migrated cells of the transwell migration assay in PANC-1 and (c) Mia PaCa-2 cells in the presence of a pH gradient. (d) Representative microscopic images of PDAC cell invasion assay, showing cells that invaded through the Matrigel-coated transwell membrane in a pH gradient. Scale bar = 100 µm. (e) Quantification of the mean number of invaded cells of the transwell invasion assay in PANC-1 and (f) Mia PaCa-2 cells in the presence of a pH gradient. (g) Effect of acidic pH_e on the Digestion Index of both PANC-1 and (h) Mia PaCa-2 control and pH_e-selected + 7.4 cells. The percentage of cells that produced invadopodia and their ECM degradation was determined by in situ zymography. The mean total invadopodia proteolytic activity was calculated as follows: Digestion Index = % of cells positive for invadopodia ECM digestion × mean pixel density of focal ECM digestion/cell. Data were reported as mean and ± SEM and analyzed using One-way ANOVA with Dunnett’s multiple comparisons test with Tukey’s multiple comparisons test for (g,h). All data shown were obtained from three independent experiments, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 5

Effects of acidic pH_e on epithelial–mesenchymal transition and proliferation markers of PDAC cell. (a) Heatmap of mRNA levels for epithelial–mesenchymal transition (EMT) markers in PANC-1 and Mia PaCa-2 cells. Columns represent each condition of the different PDAC cell lines, while rows indicate each differentially expressed gene. The value indicated is the mean value of the fold change of triplicate samples relative to control. Fold changes relative to control are visualized using a green-to-red gradient color scale. (b) mRNA expression levels of the different EMT markers and (c) proliferation (Ki67) and cell-cycle arrest (G0S2) markers in PANC-1 and Mia PaCa-2 cells subjected to acidic treatment for different periods and presented as fold change values, obtained by RT-qPCR. The effects of pH_e 6.6 treatment on EMT and proliferation marker expression were compared with control samples (dotted lines). Fold changes were quantified using the 2^−ΔΔCq method and normalized to HPRT reference gene. (d) Representative Western blot results that illustrate the effect of different times of exposure to acidic pH_e on EMT proteins in PANC-1 and Mia PaCa-2 cells. (e) Relative densiometric quantification of Western blot results, showing the abundances of N-cadherin and (f) Vimentin proteins in PANC-1 cells and (g) Vimentin protein levels in Mia PaCa-2 cells compared to control conditions after normalization with β-actin. Data were presented as mean and ± SEM and analyzed using one-way ANOVA with Dunnett’s multiple comparisons test. All data reported were obtained from three independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant. The uncropped blots are shown in Supplementary File S11—Uncropped Western Blots Membranes.

Figure 6

Differential transcriptomic profiles in PANC-1 cells in response to acidosis. (a) Heatmaps of RNA-Seq transcriptomic data relative to the differentially expressed genes (DEGs) detected comparing PANC-1 control condition against 4 days pH_e 6.6 cells and (b) PANC-1 pH_e-selected + 7.4 cells, respectively. Rows of the heatmaps represent the DEGs detected in the two comparisons, while columns are the individual biological replicates. Gene expression values are shown as gene-wise median-centered “log2(FPKM+1)” (FPKM = Fragments Per Kilobase of sequence per Million mapped reads) according to a red-to-blue color gradient. (c) MA-plots showing gene differential expression observed in PANC-1 after 4 days at pH_e 6.6 and (d) in PANC-1 pH_e-selected + 7.4 cells compared to Control. “M = log2FC” vs. “A = log2(mean counts+1)”. Downregulated genes are in red, while upregulated DEGs are in blue. Grey dots indicate non-significant gene expression changes. (e) Venn diagram summarizing the number of differentially expressed genes in PANC-1 4 days pH_e 6.6 cells vs. Control and pH_e-selected + 7.4 cells vs. Control; the number of genes deregulated in both acidic conditions is represented by the overlap between the two circles.

Figure 7

Gene ontology (GO) and gene set enrichment analysis (GSEA) of differentially expressed genes in PANC-1 cells in response to acidosis. (a) Venn plots showing overlapped dbEMT2.0 curated genes and differentially expressed genes (DEGs) in PANC-1 4 days pH_e 6.6 and (b) PANC-1 pH_e-selected cells + 7.4. (c) Bar chart of the most relevant enriched biological processes as defined in GO database and resulting from the analysis of genes downregulated in PANC-1 4 days pH_e 6.6 cells vs. Control, and (d) in PANC-1 pH_e-selected + 7.4 cells vs. Control, and of genes upregulated in (e) PANC-1 4 days pH_e 6.6 cells vs. Control, and (f) in PANC-1 pH_e-selected + 7.4 cells vs. Control. (g) RNA-Seq data of PANC-1 4 days pH_e 6.6 and (h) pH_e-selected + 7.4 cells analyzed using gene set enrichment analysis (GSEA) and presented as enrichment score (ES) plots. GSEA showed significant enrichment of cell-substrate adhesion, and migration-related gene sets in the two acidic conditions, and of proliferation-related gene set for pH_e-selected + 7.4. y-axes in GSEA plots represent the ES function, while x-axes display a red-blue color scale corresponding to the ranked list of DEGs (from the most up- to the most downregulated one, respectively) following one of the two acidic treatments. Vertical black lines above the red-blue scale in each plot refer to the position of each gene of the selected gene set along the ranked gene list as returned by the differential expression analysis of experimental data.

Figure 8

PDAC cells exposed to acidic extracellular conditions undergo a process of selection, characterized by acid-induced genetic and phenotypic alterations. This results in increased cell death due to the cytotoxic effect of low pH_e and decrease in cell-substrate adhesion, proliferation, and cell invasion. Along with the acid exposition, further genetic rewiring provides surviving cancer cells with more aggressive properties in terms of adhesion, migration, and invasion. Limited proliferative capacities are overcome when cells are acclimated to pH_e 7.4 following 1 month-long pH_e 6.6 treatment. ↓ arrow indicates downregulation, ↑ arrow indicates upregulation. The figure was created with www.Biorender.com.