Figure 3 from Acquisition of Resistance to RAS Inhibition Is Associated with the Upregulation of Macropinocytosis through Both PI3K-Dependent and -Independent Signaling
PDAC cell lines with acquired resistance to pharmacologic inhibitors of RAS exhibit enhanced macropinocytosis. A, Schematic diagram showing the experimental design for generating cell lines with acquired resistance to MRTX1133 (G12Di) and RMC7797 (RASi). B, Representative images of macropinosomes labeled with FITC–dextran (green) and nuclear DAPI stain (blue) in indicated parental or MRTX849- (G12Ci-R), MRTX1133- (G12Di-R), or RMC7797-resistant (RASi-R) KRAS-mutant PDAC cells. Images are representative of 10 fields of view analyzed in each of three independent experiments. Scale bars, 20 μm. C, Quantification of relative macropinocytic (MP) index, with each individual data point representing one field containing at least 10 analyzed cells. Data are presented as the mean ± SD of one experiment that is representative of three independent experiments. ****, ρ < 0.0001, by the unpaired Student t test, comparing against the respective parental cell line. D, Macropinocytic uptake of TMR–dextran was measured by MFI via flow cytometry in indicated parental or MRTX849- (G12Ci-R), MRTX1133- (G12Di-R), or RMC7797-resistant (RASi-R) KRAS-mutant PDAC cell lines. Data are presented as the mean ± SEM of three independent experiments. **, ρ < 0.01 and ***, ρ < 0.001 by the unpaired Student t test, comparing against the respective parental cell line. E, Representative images of macropinosomes labeled with FITC–dextran (green) and nuclear DAPI stain (blue) in indicated treatment-naïve (naïve) or RMC7797-resistant (resistant) tumor-derived KPC cells. Images are representative of 10 fields of view analyzed in each of three independent experiments. Scale bars, 20 μm. F, Macropinocytic uptake of TMR–dextran was measured by MFI via flow cytometry in indicated treatment-naïve (naïve) or RMC7797-resistant (resistant) tumor-derived KPC cell lines. Data are presented as the mean ± SEM of three independent experiments. **, ρ < 0.01 by nested two-way ANOVA. G, Relative levels of selected macropinocytosis-related proteins in PANC-1 cells following 24 or 168 hours of RASi treatment, as well as in a RASi-R cell line. Line color represents the direction of change from 24 hours RASi treatment to RASi resistance, and line transparency represents the magnitude of change. Dex, dextran; MFI, median fluorescence intensity. [A, Created in BioRender. Robb, R. (2026) https://BioRender.com/rx7i90i.]