Fig. 5: Macropinocytosis is a novel Cu supply route in mutant KRAS-driven intestinal cells.

a Macropinocytosis was visualized (left) and quantified (right) with TMR-dextran in Control and KRAS cells. EIPA served as negative control. White dashed lines indicate cell boundaries. Data are averaged for n > 167 cells and represent three independent experiments. ****P < 0.0001 (two-way ANOVA with post-hoc Bonferroni’s multiple-comparison analysis). scale = 20 μm. b, c Control and KRAS cells were treated with EIPA at the indicated doses. b Cell viability graph with IC50 values in the adjacent table. Data are presented as relative percentage (±SEM) of living cells and represent n = 3 independent replicates. c Cell lysates were collected and CCS levels were analyzed by IB and quantified (right). Data represent n = 3 (left) and n = 10 (right)independent experiments. *P = 0.0336 (unpaired two-tailed Mann–Whitney t-test). d Graphs depicting average fluorescence ratios of crisp-17 for KRAS cells treated with either DMSO (vehicle) or EIPA (20 µM). Data are averaged for n = 10 cells and represent three independent experiments. Representative fluorescence intensity images are presented (right). scale = 30 μm. e In vivo protocol used to evaluate the role of macropinocytosis in Cu-dependent KRAS tumor growth. Mice injected with KRAS IEC-6 cells were provided with a Cu-deficient diet and either deionized H₂O (diH₂O) (Group A) or diH₂O with CuSO₄ (Groups B and C) throughout the study. After two weeks, groups A and B were treated with vehicle and group C with EIPA for three weeks. Graphs depicting f, mean tumor volume over time and g mean tumor volume at the end of study (n = 6 mice). P-values between Group A and B (†, *P < 0.05; ***P < 0.001; ****P < 0.0001) and between Group B and C (§, *P < 0.05; ***P < 0.001) were determined by two-way ANOVA with post-hoc Bonferroni’s multiple-comparison analysis. h–j Representative images (left) and quantifications (right) from immunohistochemistry profiling of mouse xenograft tissues for n = 48 CCS (h), ATP7A (i), and Ki67 (j) random images from six mice per condition are depicted. scale = 250 μm. For panels a–d, f–j center values and error bars represent mean ± SEM. For panels c, d, h–j *P < 0.05; ****P < 0.0001 (unpaired two-tailed Student’s t-tests).