Fig. 5: Coadaptation is mediated by the SLIT2-ROBO1 axis.
a Representative H&E staining and IHC-P staining of ROBO1 in human PDAC primary tumours and paired liver metastatic niches displaying the importance of ROBO1 in liver metastasis (n = 35 cases, 3 fields assessed per sample). Scale bars, 100 μm. b The number of PDAC primary tumours or liver metastases expressing different ROBO1 levels according to scores on IHC-P staining is shown (n = 35 cases). c Equal amounts of Panc02Ctrl and Panc02Robo1-FL/GFP cells were mixed to prepare an intrasplenic injection model followed by metastatic niche separation and reculture. IHC-P and flow cytometry were performed to evaluate coadaptation. PG0: original cell mixture; PG1: cells derived from PG0-modelled liver metastases; PG2: cells derived from PG1-modelled liver metastases. d–e Flow cytometry to detect the cell composition of PG0, PG1 or PG2 (n = 5 technical repeats per group). f Heatmap showing the distribution of ROBO1 and SLIT2 expression in the livers of Panc02 cell mixture-modelled mice measured by IHC-P staining (n = 32 mice per group, 3 fields assessed per sample; independent experiments for each group). N: negative; W, weak; M, moderate; P, positive. g, h Mice treated with ROBO1 neutralizing antibody were modelled with a Panc02 cell mixture before IHC-P staining was performed (n = 32 mice per group, 3 fields assessed per sample; independent experiments for each group). N: negative; W, weak; M, moderate; P, positive. i The strategy for exploring the coadaptation mechanism mediated by SLIT2-ROBO1 in liver metastatic niches. A mixture of equal amounts of Kpc1199Robo1-FL/GFP cells and Kpc1199ΔRobo1/mCherry cells (Mix-I) or Kpc1199Robo1-FL/GFP cells and Kpc1199CTRL/mCherry cells (Mix-II) was intrasplenically injected into Slit2/CKO or CTRL mice for further examination. j, k Representative IF staining of a Kpc1199 cell mixture showing the formation of liver metastatic niches indicating coadaptation (n = 5 mice per group, 3 fields assessed per sample, mean ± SEM.; two-tailed unpaired t test). GFP, green; mCherry, red; DAPI, blue. Scale bars, 100 μm. l Flow cytometry detection of the ratio of two types of Kpc1199 cells in separated liver metastatic niches. Source data are provided in the Source Data file.
