Fig 2: EphA2pos cells are similarly represented in CRC specimens and matched PDOs, and they persist over PDO passaging. Patient-derived organoids (PDOs) were obtained from seven CRC specimens and characterized for their expression of mRNA markers immediately after disaggregation (p0) and after two to four passages (p2, p3, p4). (a) Representative micrographs show PDOs growing over time after disaggregation, forming convoluted structures. Scale bar: 200 μm (b,c). Graphs showing the relative expression levels of CDX2, CK20, CD44, and EpCAM mRNA, as detected by QRT-PCR, in PDO cultures at passages zero (reference: p0), two (p2), three (p3), and four (p4). The average of triplicate experiments for each passage is reported. Values are expressed as fold change over control (p0, set at 1). No significant changes in the expression of the markers were recorded, except where indicated by the asterisk (*). *p < 0.05 vs. passage 0. (d) Representative dot plots showing the percentage of EphA2-positive cells/live cells evaluated by flow cytometry in PDO#4 at p0 and after serial passaging (p3 and p5). Gating was determined based on staining with an irrelevant isotype antibody. (e) The histogram shows the percentage of EphA2-positive cells at p0, p3, and p5 in all seven PDO cultures. No statistically significant differences were observed at p0, p3, or p5 (p > 0.05). Statistics: *p < 0.05; **p < 0.01. Absence of asterisk denotes no statistically significant difference.

Fig 3: OXA treatment increases the levels of EphA2 in CRC PDOs. (a) The levels of EphA2 total protein after OXA treatment were detected by ELISA at the indicated times. Please note that differences were statistically significant at 24 and 48 h After treatment. (b) Western blotting of whole-cell lysates from PDO#4, which were transfected with scrambled or EphA2-directed siRNA, treated with vehicle or OXA, and stained with an anti-EphA2 antibody. Tubulin was used as a loading control. (c) Representative micrographs of PDO#4 treated with scrambled or EphA2 RNAi in the presence or absence of OXA. (d) Quantitative evaluation of formed organoids/well). Statistics: *p < 0.05; **p < 0.01. Absence of asterisk denotes no statistically significant difference.

Fig 4: EpHA2pos cells persist in oxaliplatin-treated CRC PDOs. (a) PDOs one to seven were treated with Ctrl (saline) or 5 μmol/L OXA for 96 h (with drug washout at 24 h). (a) The number of newly formed organoids after disaggregation and at the end of the treatment was recorded (and defined as OFA; see text). (b) Left panel. The average diameter of the formed organoids at the end of the treatment was assessed with ImageJ on phase contrast images. Right panel. Percentage of live cells evaluated by flow cytometry in disaggregated PDO treated with Ctrl or OXA. (c) Representative phase-contrast images of PDO #4 treated with 5 μM OXA for the indicated days, then fixed with PFA and stained with an anti-EpHA2 antibody at each indicated time point. The relative position of the PDOs is indicated by dashed circles. (d) Median fluorescence intensity (MFI) for EphA2-positive cells in PDOs #one to seven treated as in (c), calculated after selecting positive cells as ROI. (e) Histogram reporting the levels of EphA2 in the whole cell lysate (WCL) of PDOs harvested at the indicated times after OXA treatment, detected by ELISA. (f) Graph reporting the organoid-forming ability (reported as OG/well) of PDOs #one to seven treated with OXA for 21 days (g). Percentage of EphA2-positive cells/live cells after 21 days of oxaliplatin treatment, assessed by flow cytometry (a–g) Mean +SEM of triplicate measurements. (h) Linear correlation analysis between the number of EphA2-positive cells and the formed organoids per well, after chronic Ctrl and OXA treatment. Data from quadruplicate measurements (r = 0.3345 and p = 0.0073). Statistics: *p < 0.05; **p < 0.01. Absence of asterisk denotes no statistically significant difference.

Fig 5: OXA treatment increases the Ser897 phosphorylation of EphA2 in an MK2-dependent way. (a) Western blotting with an EphA2 antibody of whole cell lysate (WCL) from PDO#4, treated as indicated with 5 μmol/L OXA, and subjected to Zn-Phos-Tag SDS-PAGE gel separation. (b) ELISA assay to detect the Ser897-EphA2 levels in time after OXA treatment. (c) Levels of Ser-897 EphA2 after treatment with the MK2 inhibitor PF-3644022, in the presence of vehicle or OXA, detected by ELISA (b,c) Mean ± SEM of quadruplicate detections. (d) Representative micrographs of PDO #4 treated with vehicle or PF-3644022 in the presence or absence of OXA. (e) Quantitative evaluation of organoid-forming ability (OG/well) for the PDO#4 treated as in (d). Mean ± SEM of sextuplicate measurements. (f) The phosphorylation of Ser897-EphA2 is common to OXA-resistant PDOs. (f) ELISA assay to detect the levels of Ser897-EphA2 in the seven PDOs treated with OXA for 12 h. (g) Treatment with the MK2 inhibitor PF-3644022 blunts PDO resistance to OXA. The number of OG/well was evaluated after the indicated in the OXA-resistant PDOs (f,g) Mean ± SEM of triplicate measurements. Statistics: *p < 0.05; **p < 0.01. Absence of asterisk denotes no statistically significant difference. Please note that in (g) the statistics are reported for OXA vs. PF-364402 + OXA.